Microfluidic devices

ABSTRACT

The present invention provides novel microfluidic substrates and methods that are useful for performing biological, chemical and diagnostic assays. The substrates can include a plurality of electrically addressable, channel bearing fluidic modules integrally arranged such that a continuous channel is provided for flow of immiscible fluids.

RELATED APPLICATIONS

This patent application is a continuation of U.S. NonprovisionalApplication No. 15/886,212, filed Feb. 1, 2018, which is a continuationof U.S. Nonprovisional Application No. 15/480,739, filed Apr. 6, 2017,which is a continuation of U.S. Nonprovisional Application No.13/779,943, filed Feb. 28, 2013 (now U.S. Pat. No. 9,981,230), which isa continuation application of U.S. Nonprovisional Application No.11/803,104, filed May 11, 2007, which claims priority to, and thebenefit of, U.S. Provisional Application Nos. 60/799,833 filed on May11, 2006; 60/799,834 filed on May 11, 2006; 60/808,614 filed on May 25,2006; 60/815,097 filed on Jun. 19, 2006; 60/819,733 filed on Jul. 7,2006; 60/819,734 filed on Jul. 7, 2006; 60/841,716 filed on Sep. 1,2006; 60/843,374 filed on Sep. 8, 2006; 60/833,151 filed on Jul. 24,2006; 60/834,987 filed on Jul. 31, 2006; 60/837,871 filed on Aug. 14,2006; 60/837,695 filed on Aug. 14, 2006; 60/843,327 filed on Sep. 8,2006; 60/856,540 filed on Nov. 3, 2006; 60/856,440 filed on Nov. 3,2006; 60/874,561 filed on Dec. 12, 2006; 60/858,279 filed on Nov. 8,2006; 60/858,278 filed on Nov. 8, 2006; 60/874,640 filed on Dec. 12,2006; 60/860,665 filed on Nov. 22, 2006; 60/873,766 filed on Dec. 8,2006; 60/876,209 filed on Dec. 20, 2006; 60/899,258 filed on Feb. 2,2007; 60/903,153 filed on Feb. 23, 2007; 60/904,293 filed on Feb. 28,2007; 60/920,337 filed on Mar. 26, 2007. The contents of each of theseapplications are incorporated herein by reference in their entirety.

FIELD OF INVENTION

The present invention generally relates to systems and methods for theformation and/or control of fluidic species, and articles produced bysuch systems and methods. More particularly, the present inventionrelates to the development of high throughput microfluidic devices forprecision fluid handling and use of such systems in various biological,chemical, or diagnostic assays.

BACKGROUND

The manipulation of fluids to form fluid streams of desiredconfiguration, discontinuous fluid streams, droplets, particles,dispersions, etc., for purposes of fluid delivery, product manufacture,analysis, and the like, is a relatively well-studied art. For example,highly monodisperse gas bubbles, less than 100 microns in diameter, havebeen produced using a technique referred to as capillary flow focusing.In this technique, gas is forced out of a capillary tube into a bath ofliquid, where the tube is positioned above a small orifice, and thecontraction flow of the external liquid through this orifice focuses thegas into a thin jet which subsequently breaks into equal-sized bubblesvia a capillary instability. A similar arrangement can be used toproduce liquid droplets in air.

Microfluidic systems have been described in a variety of contexts,typically in the context of miniaturized laboratory (e.g., clinical)analysis. Other uses have been described as well. For example,International Patent Application Publication No. WO 01/89788 describesmulti-level microfluidic systems that can be used to provide patterns ofmaterials, such as biological materials and cells, on surfaces. Otherpublications describe microfluidic systems including valves, switches,and other components.

Precision manipulation of streams of fluids with microfluidic devices isrevolutionizing many fluid-based technologies. Networks of smallchannels are a flexible platform for the precision manipulation of smallamounts of fluids. The utility of such microfluidic devices dependscritically on enabling technologies such as the microfluidic peristalticpump, electrokinetic pumping, dielectrophoretic pump or electrowettingdriven flow. The assembly of such modules into complete systems providesa convenient and robust way to construct microfluidic devices. However,virtually all microfluidic devices are based on flows of streams offluids; this sets a limit on the smallest volume of reagent that caneffectively be used because of the contaminating effects of diffusionand surface adsorption. As the dimensions of small volumes shrink,diffusion becomes the dominant mechanism for mixing leading todispersion of reactants; moreover, surface adsorption of reactants,while small, can be highly detrimental when the concentrations are lowand volumes are small. As a result current microfluidic technologiescannot be reliably used for applications involving minute quantities ofreagent; for example, bioassays on single cells or library searchesinvolving single beads are not easily performed. An alternate approachthat overcomes these limitations is the use of aqueous droplets in animmiscible carrier fluid; these provide a well defined, encapsulatedmicroenvironment that eliminates cross contamination or changes inconcentration due to diffusion or surface interactions. Droplets providethe ideal microcapsule that can isolate reactive materials, cells, orsmall particles for further manipulation and study. However, essentiallyall enabling technology for microfluidic systems developed thus far hasfocused on single phase fluid flow and there are few equivalent activemeans to manipulate droplets requiring the development of droplethandling technology. While significant advances have been made indynamics at the macro- or microfluidic scale, improved techniques andthe results of these techniques are still needed. For example, as thescale of these reactors shrinks, contamination effects due to surfaceadsorption and diffusion limit the smallest quantities that can be used.Confinement of reagents in droplets in an immiscible carrier fluidovercomes these limitations, but demands new fluid-handling technology.

Furthermore, the underlying physics of the influence of electric fieldson fluids is well known. The attractive and repulsive forces produced byan electric field on positive or negative charges give rise to theforces on charged fluid elements, the polarization of non-polarmolecules, and the torque on polar molecules which aligns them with thefield. In a non-uniform field, because the force on the positivelycharged portion of the distribution is different than the force on thenegatively charged portion, polar molecules will also experience a netforce toward the region of higher field intensity. In the continuumlimit, the result is a pondermotive force in the fluid. In the limit ofhigh droplet surface tension, it is useful to describe the netpondermotive force on a droplet as if it were a rigid sphere:

F=qE+2π

(ε_(m))r ³

(K)∇E ²,

where the first term is the electrophoretic force on the droplet (q isthe net droplet charge and E is the electric field), and the second termis the dielectrophoretic force (r is the radius of the sphere, R(K) isthe real part of the Clausius-Mossotti factor

K=(ε*_(p)−ε*_(m))/(ε*_(p)+2ε*_(m)),

and ε*_(p) and ε*_(m) are the complex permittivities of the droplet andcarrier fluid).

Although utility of electrophoretic control of droplets is great, itdoes have significant limitations. First, the charging of droplets isonly effectively accomplished at the nozzle. Second, the discharge pathrequired to eliminate screening effects also discharges the droplets.Third, finite conductivity of the carrier fluid, however small, willeventually discharge the droplets. Therefore, once the droplet isformed, there is essentially only one opportunity to perform anypondermotive function which relies on the droplet's charge density (suchas coalescing oppositely charged droplets through their mutual Coulombicattraction, or electrophoretically sorting a droplet), and that functioncan only be performed as long as sufficient charge has not leaked off ofthe droplet.

Thus, it would be desirable to develop an electrically addressableemulsification system that combines compartmentalization and electricalmanipulation, which allows for multi-step chemical processing, includinganalysis and sorting, to be initiated in confinement with exquisitetiming and metering precision, for use in a variety of chemical,biological, and screening assays, in which the cost and time to performsuch assays would be drastically reduced. It would also be desirable todevelop a device using dielectrophoretic force (which does not rely oncharge density) to manipulate droplets so that more than one electricalpondermotive function can be carried out following a significantly longdelay from droplet formation.

SUMMARY OF THE INVENTION

The present invention provides substrates having individual fluidhandling modules that can be combined into fluid processing systems soas to perform multi-step processing of isolated components, which isessential to perform biological, chemical and diagnostic applications,quickly, effectively and inexpensively. Using principles based on theelectrical manipulation of droplets, the microfluidic substrates of thepresent invention can encapsulate reagents into droplets, which can becombined, analyzed, and sorted.

The present invention provides a microfluidic substrate. The substratecan include a plurality of Microfluidic modules integrally arranged witheach other so as to be in fluid communication. The substrate caninclude, for example, (i) at least one inlet module having at least oneinlet channel adapted to carry at least one dispersed phase fluid, (ii)at least one main channel adapted to carry at least one continuous phasefluid, wherein the inlet channel is in fluid communication with the mainchannel at a junction, wherein the junction includes a fluidic nozzledesigned for flow focusing such that the dispersed phase fluid isimmiscible with the continuous phase fluid and forms a plurality ofhighly uniform, monodisperse droplets in the continuous phase fluid. Theflow of the dispersed phase and continuous phase can be pressure driven,for example. The dispersed phase (e.g. droplets) can be neutral or haveno charge and these droplets can be manipulated (e.g., coalesced,sorted) within a electric field in the continuous phase fluid.

The inlet module can further include at least one self-aligning fluidicinterconnect apparatus to connect the inlet channel to a means forintroducing a sample fluid to the channel, wherein the apparatus forms aradial seal between the microfluidic substrate and the means forintroducing sample. The means can include, for example, a well orreservoir, which can be temperature controlled. The well or reservoircan optionally include an acoustic actuator.

The microfluidic substrate can include one or more additional modules,including but not limited to, coalescence module, detection module,sorting module, collection module, waste module, delay module (e.g.,heating and cooling modules), droplet spacing module, mixing module,UV-release module, division module and/or reordering module. Thesemodules are in fluid communication with the main channel. There may bezero, one, or more of each of the modules.

The substrate can further include at least one coalescence moduledownstream from and in fluid communication with the inlet module via themain channel including a coalescence apparatus, wherein two or moredroplets passing there through are coalesced to form a nanoreactor.

The substrate can further include at least one detection moduledownstream from and in fluid communication with the coalescence module.The detection module can include, for example, a detection apparatus forevaluating the contents and/or characteristics of the nanoreactor. Thedetection apparatus can include an optical or electrical detector.

The substrate can further include a sorting module downstream from andin fluid communication with the detection module. The sorting module caninclude, for example, a sorting apparatus adapted to direct thenanoreactor into or away from a collection module in response to thecontents or characterization of the nanoreactor evaluated in thedetection module. The channels in the sorting module can include anasymmetric bifurcation geometry or an asymmetric bifurcation flow.

The coalescence apparatus and the sorting apparatus can include one ormore electrodes, or a patterned electrically conductive layer, which arecapable of generating an electric field. The electrodes can be made fromelectrically conductive materials, and can be integrally contained inone or more channels isolated from the main and inlet channels of thesubstrate. The electrically conductive materials can be metal alloycomponents or organic materials. The electrically conductive materialcan be an epoxy resin including one or more electrically conductiveparticles. The electrically conductive particles can be silverparticles.

The coalescence module can further include an expanded portion of themain channel between the electrodes to bring successive droplets intoproximity, whereby the paired droplets are coalesced within the electricfield. The coalescence module can further include a narrowed portion ofthe main channel to center droplets within the main channel prior to theexpanded portion of the main channel between the electrodes.

The channels of the microfluidic substrate can be coated with ananti-wetting or blocking agent for the dispersed phase. The anti-wettingor blocking agent can include, for example, a silica primer layerfollowed by a perfluoroalkylalkylsilane compound, an amorphous solubleperfloropolymer, BSA, PEG-silane or fluorosilane. The channels of themicrofluidic substrate can include well-like indentations to slow, stopor react contents of droplets.

The substrate can further include a collection module connected to ameans for storing a sample from the substrate and a waste moduleconnected to a means for collecting a sample discarded from thesubstrate. The means can be a well or reservoir, which can betemperature controlled.

The substrate can further include a delay module in fluid communicationwith the main channel downstream of the coalescence module and upstreamof the detection module. The delay module can be a delay line,serpentine channel, a buoyant hourglass, or an off-chip volume.Preferably, a serpentine channel is used to time delays less than 1hour. Preferably, an off-chip volume is used to time delays longer than1 hour. The delay module can further include heating and coolingregions.

The substrate can further include a mixing module in fluid communicationwith the main channel downstream of the coalescence module and upstreamof the detection module.

The substrate can further include a UV-release module in fluidcommunication with the main channel downstream of the inlet module andupstream of the coalescence module.

The substrate can further include a droplet spacing module in fluidcommunication with the main channel downstream of the inlet module toallow appropriate droplets to come with proximity for coalescence.

The continuous phase used in the channels of the microfluidic substratecan be a non-polar solvent such as, for example, a fluorocarbon oil. Thecontinuous phase can further include one or more additives such as asurfactant or fluorosurfactant in order to stabilize the droplets. Thefluorosurfactant can be a perfluorinated polyether, for example.

The dispersed phase of the microfluidic substrate can include a libraryof droplets of the same or different sizes (i.e., an emulsion stream) ora continuous aqueous stream. The library of droplets can include, forexample, a biological or chemical material such as tissues, cells,particles, proteins, antibodies, amino acids, nucleotides, smallmolecules, and pharmaceuticals. The biological/chemical material caninclude a label such as a DNA tag, dye, a quantum dot or a radiofrequency identification tag. The library of library of droplets caninclude a label such as a change in viscosity, a change in opacity, achange in volume, a change in density, a change in pH, a change intemperature, a change in dielectric constant, a change in conductivity,a change in the amount of beads present in the droplets, a change in theamount of flocculent in the droplets, a change in the amount of aselected solvent within the droplets or the change in the amount of anymeasurable entity within the droplets, or combinations thereof. A labelcan be detected by fluorescence polarization, fluorescence intensity,fluorescence lifetime, fluorescence energy transfer, pH, ionic content,temperature or combinations thereof.

The present invention also provides a microfluidic substrate including,for example, (i) at least one inlet module having at least one inletchannel adapted to carry at least one dispersed phase fluid; (ii) atleast one main channel adapted to carry at least one continuous phasefluid, wherein the inlet channel is in fluid communication with the mainchannel at a junction, wherein the junction includes a fluidic nozzledesigned for flow focusing such that the dispersed phase fluid isimmiscible with the continuous phase fluid and forms a plurality ofhighly uniform, monodisperse droplets in the continuous phase fluid;(iii) at least one nanoreactor division module downstream from the inletmodule wherein the main channel is divided into at least two divisionchannels and the nanoreactor is split into at least two daughternanoreactors; (iv) at least one second inlet channel adapted to carry atleast one second dispersed phase fluid wherein the inlet channel is influid communication with at least one of the divisional channels at ajunction, wherein the junction includes a fluidic nozzle designed forflow focusing such that the second dispersed phase fluid is immisciblewith the continuous phase fluid and forms a plurality of highly uniform,monodisperse droplets in the continuous phase fluid; (v) at least onecoalescence module downstream from and in fluid communication with theinlet module via the main channel including a coalescence apparatus,wherein at least one droplet from step (ii) and at least one dropletfrom step (iv) passing there through are coalesced; (vi) at least onereorder module downstream from the dividing module such that thedaughter nanoreactors from the division channel are reordered inproximity but not coalesced; and (vii) at least one detection moduledownstream from the reorder module, the detection module including adetection apparatus for evaluating the contents or characteristics of atleast one of nanoreactors or droplets in proximity.

The microfluidic substrate can also include a sorting module inproximity to and in fluid communication with the detection module, thesorting module including a sorting apparatus adapted to direct thedroplet or nanoreactor into or away from a collection module in responseto the contents or characterization of the droplet or nanoreactorevaluated in the detection module.

The detection module can evaluate the contents of two nanoreactors ordroplets in proximity and the sorting module can direct the droplets ornanoreactors into or away from a collection module in response to theratio of the contents or characterization of the droplets ornanoreactors evaluated in the detection module.

The present invention also provides a method of producing microfluidicsubstrate including, for example, (i) providing a base plate, whereinthe base plate comprises a flat surface; (ii) providing a masterincluding the pattern of the channels and electrodes of a microfluidicsubstrate; (iii) providing a molding cavity, wherein the molding cavitycomprises an opening for molding an elastomeric substrate; (iv)assembling the base plate, master and molding cavity, such that themaster is placed between the base plate and molding cavity and whereinthe master pattern is located directly under and aligned to the openingfor molding an elastomeric substrate; (v) providing a top platecontaining one or more sliding molding pins used to form one or morefluid and/or electrical interconnects; (vi) assembling the top plateonto the molding cavity of step d, such that the sliding molding pinscontact points on the pattern of channels and electrodes on the master;(vi) introducing a liquid elastomeric polymer into the opening on themolding cavity such that it contacts the master; (vii) solidifying theelastomeric polymer within the molding cavity; (viii) removing thesolidified elastomeric polymer substrate from the top plate, bottomplate and molding cavity assembly; and (ix) bonding the solidifiedelastomeric polymer substrate to compatible polymeric or non-polymericmedia. The sliding molding pins can be surrounded by an elastomericsleeve. The present invention also provides a microfluidic deviceproduces by the methods provided.

The master is generated by photolithography, photolithography andconverted to a durable metal master, micromachining or by rapidprototyping methods such as stereolithography. The master can be asilicon or glass substrate patterned with photoresist. Preferably, themaster is a silicon or glass substrate patterned with SU-8.

The elastomeric polymer can be a silicone elastomeric polymer.Preferably, the silicone elastreric polymer is polydimethylsiloxane. Theelastomeric polymer can be solidified by curing. The elastomeric polymercan be treated with high intensity oxygen or air plasma to permitbonding to the compatible polymeric or non-polymeric media. Thepolymeric and non-polymeric media can be glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, orepoxy polymers.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying drawings, which areschematic and are not intended to be drawn to scale. In the drawings,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For the purposes of clarity, not everycomponent is labeled in every drawing, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe drawings:

FIG. 1 is an schematic illustrating the interacting modules of amicrofluidic device of the present invention.

FIGS. 2A-B show dual and single oil versions of the nozzle concept usinga small ferrule for the nozzle section. FIGS. 2C-D show the same nozzlesmade directly out of small bore tubing (the “nozzle” runs the entirelength of the tubing).

FIG. 3 shows the expansion of the nozzle ferrule concept shown in FIGS.2A and 2B.

FIG. 4 shows the expansion of the nozzle section contained in theferrule.

FIG. 5A shows the operation of the nozzle in Aspiration Mode and 5Bshows the operation of the nozzle in Injection Mode.

FIGS. 6A-D show a reservoir based sample emulsification. FIG. 6A showswhere the well is initially filled with a fluid with lower density thanthe sample to be introduced. 6B-6D show a more dense sample dropletbeing introduced to the well and settling to the bottom of the well andbeing transported to the nozzle. 6E shows that the oil lines would beginflowing at their prescribed rates, while the collection or waste portwould begin withdrawing at a rate.

FIGS. 7A-E illustrate a sample introduction when the sample is lessdense than the fluid in the sample port, which is an alternative schemeused to introduce samples that are less dense than the oil used toemulsify the sample. 7A shows that the sample port could be filled withthe emulsification oil through backflow from the nozzle prior tointroduction of the sample. 7B-7D show a sample tip connected to a pumpcapable of driving the sample into the device. The pump is started up asthe tip is inserted into the device, injecting sample in to the port. 7Eshows that the oil lines would begin flowing at their prescribed rates,while the collection or waste port would begin withdrawing at a rate.

FIG. 8 illustrates a nozzle that formed directly into the fitting usedto connect the collection syringe to a syringe tip (e.g. capillarytubing) in order to create a monodisperse emulsion directly from alibrary well. Step 1 shows the aspiration of the sample can beaccomplished by running the collection syringe in withdrawal mode at aflow rate (Q3) above the flow rate of the two oil syringes. Step 2 showsthe appropriate volume of sample loaded into the capillary tubing, andthe capillary tubing would be removed from the sample well, an airbubble, and possibly a cleaning solution would be aspirated. Step 3shows when almost all of the sample has been emulsified, the collectionsyringe withdrawal rate would either be reduced below the oil flowrates, stopped, or set to infuse at some nominal rate.

FIGS. 9A-C illustrate a two phase system where the reagent is injectedon top of the 2nd, immiscible phase. (FIG. 9A) During injection, priorto transition from 1st phase to 2nd phase. (FIG. 9B) 2nd phase justentering the transfer lines. (FIG. 9C) 2nd phase has completely filledthe transfer line and pushed the entire volume of reagent through thesystem.

FIG. 10 illustrates sandwiching an ultra-small volume of fluid (i.e.,sub-nanoliter) between two solutions having different densities.

FIG. 11 illustrates possible interconnect designs for use with PDMSdevices.

FIG. 12 illustrates self-alignment of fluidic interconnect

FIG. 13 illustrates the interconnects needed for each tube molded into asingle monolithic self-aligned part.

FIG. 14 shows a schematic of a molding tool based on this concept. Thepins (orange) are captured within an elestomeric molded sleeve and acompression plate made from a rigid backer plate and foam rubber is usedto apply gentle even pressure to the pins and generate the force neededto make the pins uniformly contact the master.

FIG. 15 is a schematic diagram of an improved coalescence module thatshows an optional small constriction (neckdown) just before thisexpansion can be used to better align the droplets on their way into thecoalescence point.

FIG. 16 illustrates that fluorescence polarization (FP) measures thetumbling rate of a compound in solution and is a function of it's volume(in most cases, volume is correlated with MW)

FIG. 17 shows the fluorescence polarization of three differentcompounds. Results of reading polarization in 18,000 drops containing 3distinct species (FC, BTFC, and BTFC bound to SA). Ideal for readingresults of drug screening assays, protein interactions, or DNAhybridization.

FIG. 18A illustrates encoding a liquid solution using both overallfluorescence polarization and overall dye intensity within droplets;FIG. 18B shows that multiple colors of fluorescence polarization and FIincreases the number of possible labels. Ten intensity levels with tenfluorescence polarization levels on two colors yields 10,000 labels.

FIG. 19 illustrates FPcoding using dyes having different fluorescencelifetimes. These were made one element at a time, stored in a singlesyringe overnight and then loaded back on chip. The codes were made byusing a ratio of two different dyes, one with a short lifetime and hencehigh FP and one with a long lifetime and correspondingly low FP. Themixtures have intermediate FP signals. The intensity is tuned bycontrolling the overall concentration of the two dyes.

FIGS. 20A-20D illustrate the sorting and/or splitting of droplets inaccordance with another embodiment of the invention

FIGS. 21A-F shows the possible flow geometries used in an asymmetricsorting application.

FIGS. 22A-E show the possible electrode geometries used in an asymmetricsorting application. FIG. 22A shows the design using sharp tippedelectrodes. FIG. 22B shows broad tipped electrodes to increase theinteraction time between the droplets and the electric field (the tipscould be many drop diameters long). FIG. 22C shows electrodes straddlingthe collection line. FIG. 22D shows electrodes on opposite sides of themain channel. FIG. 22E shows an Asymmetric Electrode Pair (the asymmetrymay be present on any of the other electrode pair layouts as well).

FIG. 23 shows a schematic of a device that split droplets, performsdifferent experiments on the two daughter droplets and then reorders sothat they pass sequential through the detector

FIG. 24A, shows geometric parameters defining the obstacle matrix. FIG.24B shows three fluid streams. FIG. 24C shows a particle with a radiusthat is larger than lane 1 follows a streamline passing through theparticle's center (black dot).

FIG. 25 shows high-resolution separation of fluorescent microsphereswith diameters of 0.80 um (green), 0.90 um (red), and 1.03 um (yellow),with a matrix of varying gap size.

FIG. 26 is a schematic illustrating the separation by deterministiclateral displacement in an array of microposts, with an example rowshift fraction of one-third.

FIG. 27 shows a dideoxynucleotide sequencing on a microfabricated chip.Shown is one embodiment for a DNA sequencing chip design. Template DNAand primers are combined at step ‘add 1’ and the reaction is incubatedat 95° C. for a hot start (position 1). The reaction then cycles 20-30times (position 2) before the addition of SAP and Exol at ‘add 2.’ Thereaction is incubated at 37° C. for a pre-defined time-period and thenthe SAP and Exol enzymes are inactivated at 95° C. (position ‘4’). TheSAP/Exol procedure degrades nucleotides and single-stranded DNA(primers) remaining after PCR. The universal sequencing primers, ddNTPsand buffers are added at ‘add 3,’ and the PCR sequencing reaction isallowed to proceed at position ‘5.’ The final reaction product iscollected and can be stored off-chip.

FIG. 28A, shows a schematic of the TempliPhi amplification process usingrolling circle amplification. FIG. 28B illustrates a transcriptionmediated reaction. FIG. 28C illustrates strand-displacementamplification. FIG. 28D shows a schematic diagram of helicase-dependentamplification.

FIGS. 29A-D illustrate emulsion-based sample preparation, samplepreparation and DNA sequencing. Random libraries of DNA fragments aregenerated by shearing an entire genome and isolating single DNAmolecules by limiting dilution. In FIG. 29A, genomic DNA is isolated,fragmented, ligated to adapters and separated into single strands. FIG.29B shows microscope photograph of emulsion showing droplets containinga bead and empty droplets. FIG. 29C shows a scanning electron micrographof a portion of a fiber-optic slide, showing fiber-optic cladding andwells before bead deposition. FIG. 29D shows the sequencing instrumentand its sub-systems.

FIG. 30 shows one method for isolating antibodies on a microfluidicdevice.

FIG. 31 shows an alternate method for isolating antibodies on amicrofluidic device.

FIG. 32 shows the method of the present invention for isolatingantibodies on the microfluidic device. The right panel is a diagram ofindividual steps proposed to amplify signal of interacting antibody andantigen. The left panel is a schematic as would be designed for a chipto be used on microfluidic device.

FIG. 33 shows the genetic selection for full length antibody clones. Agenetic selection can be used to enrich for full-length antibody clonesby transforming E.coli and selecting for clones able to grow on mediumin which a suitable sugar is the only carbon source.

FIG. 34 is a schematic representation of a multi-step chip according tothe invention. 1^(st) stage: droplets are sent into C2, the collectionport; 2^(nd) stage: 1^(st) stage emulsion collected into C2 isreinjected back into the chip and merged with the droplets formed in thesecond stage nozzle.

FIG. 35 shows karyotyping using spectral probes that allow all 23 pairsof human chromosomes to be seen at one time, with each pair ofchromosomes painted in a difference fluorescent color.

FIG. 36 shows the future BioBased Economy with six building blocks basedon renewable 30 biomass.

FIG. 37 shows the 8 non-renewable building blocks based on petroleum.

DETAILED DESCRIPTION

The microfluidic devices and methods of use described herein are basedon the creation and electrical manipulation of aqueous phase dropletscompletely encapsulated by an inert immiscible oil stream. Thiscombination enables precise droplet generation, highly efficient,electrically addressable, droplet coalescence, and controllable,electrically addressable single droplet sorting. The microfluidicdevices include one or more channels and modules. A schematicillustrating one example of interacting modules of a microfluidicsubstrate is shown in FIG. 1. The integration of these modules is anessential enabling technology for a droplet based, high-throughputmicrofluidic reactor system.

The microfluidic devices of the present invention can be utilized fornumerous biological, chemical, or diagnostic applications, as describedin further detail herein.

Substrates

The microfluidic device of the present invention includes one or moreanalysis units. An “analysis unit” is a microsubstrate, e.g., amicrochip. The terms microsubstrate, substrate, microchip, and chip areused interchangeably herein. The analysis unit includes at least oneinlet channel, at least one main channel, at least one inlet module, atleast one coalescence module, and at least one detection module. Theanalysis unit can further includes one or more sorting modules. Thesorting module can be in fluid communication with branch channels whichare in fluid communication with one or more outlet modules (collectionmodule or waste module). For sorting applications, at least onedetection module cooperates with at least one sorting module to divertflow via a detector-originated signal. It shall be appreciated that the“modules” and “channels” are in fluid communication with each other andtherefore may overlap; i.e., there may be no clear boundary where amodule or channel begins or ends. A plurality of analysis units of theinvention may be combined in one device. The analysis unit and specificmodules are described in further detail herein.

The dimensions of the substrate are those of typical microchips, rangingbetween about 0.5 cm to about 15 cm per side and about 1 micron to about1 cm in thickness. A substrate can be transparent and can be coveredwith a material having transparent properties, such as a glasscoverslip, to permit detection of a reporter, for example, by an opticaldevice such as an optical 25 microscope. The material can be perforatedfor functional interconnects, such as fluidic, electrical, and/oroptical interconnects, and sealed to the back interface of the device sothat the junction of the interconnects to the device is leak-proof. Sucha device can allow for application of high pressure to fluid channelswithout leaking.

A variety of materials and methods, according to certain aspects of theinvention, can be used to form any of the described components of thesystems and devices of the invention. In some cases, the variousmaterials selected lend themselves to various methods. For example,various components of the invention can be formed from solid materials,in which the channels can be formed via molding, micromachining, filmdeposition processes such as spin coating and chemical vapor deposition,laser fabrication, photolithographic techniques, etching methodsincluding wet chemical or plasma processes, and the like. See, forexample, Scientific American, 248:44-55, 1983 (Angell, et al). At leasta portion of the fluidic system can be formed of silicone by molding asilicone chip. Technologies for precise and efficient formation ofvarious fluidic systems and devices of the invention from silicone areknown. Various components of the systems and devices of the inventioncan also be formed of a polymer, for example, an elastomeric polymersuch as polydimethylsiloxane (“PDMS”), polytetrafluoroethylene (“PTFE”)or Teflon®, or the like.

The channels of the invention can be formed, for example by etching asilicon chip using conventional photolithography techniques, or using amicromachining technology called “soft lithography” as described byWhitesides and Xia, Angewandte Chemie International Edition 37, 550(1998). These and other methods may be used to provide inexpensiveminiaturized devices, and in the case of soft lithography, can providerobust devices having beneficial properties such as improvedflexibility, stability, and mechanical strength. When optical detectionis employed, the invention also provides minimal light scatter frommolecule, cell, small molecule or particle suspension and chambermaterial.

Different components can be formed of different materials. For example,a base portion including a bottom wall and side walls can be formed froman opaque material such as silicone or PDMS, and a top portion can beformed from a transparent or at least partially transparent material,such as glass or a transparent polymer, for observation and/or controlof the fluidic process. Components can be coated so as to expose adesired chemical functionality to fluids that contact interior channelwalls, where the base supporting material does not have a precise,desired functionality. For example, components can be formed asillustrated, with interior channel walls coated with another material.Material used to form various components of the systems and devices ofthe invention, e.g., materials used to coat interior walls of fluidchannels, may desirably be selected from among those materials that willnot adversely affect or be affected by fluid flowing through the fluidicsystem, e.g., material(s) that is chemically inert in the presence offluids to be used within the device.

Various components of the invention when formed from polymeric and/orflexible and/or elastomeric materials, and can be conveniently formed ofa hardenable fluid, facilitating formation via molding (e.g. replicamolding, injection molding, cast molding, etc.). The hardenable fluidcan be essentially any fluid that can be induced to solidify, or thatspontaneously solidifies, into a solid capable of containing and/ortransporting fluids contemplated for use in and with the fluidicnetwork. In one embodiment, the hardenable fluid comprises a polymericliquid or a liquid polymeric precursor (i.e. a “prepolymer”). Suitablepolymeric liquids can include, for example, thermoplastic polymers,thermoset polymers, or mixture of such polymers heated above theirmelting point. As another example, a suitable polymeric liquid mayinclude a solution of one or more polymers in a suitable solvent, whichsolution forms a solid polymeric material upon removal of the solvent,for example, by evaporation. Such polymeric materials, which can besolidified from, for example, a melt state or by solvent evaporation,are well known to those of ordinary skill in the art. A variety ofpolymeric materials, many of which are elastomeric, are suitable, andare also suitable for forming molds or mold masters, for embodimentswhere one or both of the mold masters is composed of an elastomericmaterial. A non-limiting list of examples of such polymers includespolymers of the general classes of silicone polymers, epoxy polymers,and acrylate polymers. Epoxy polymers are characterized by the presenceof a three-membered cyclic ether group commonly referred to as an epoxygroup, 1,2-epoxide, or oxirane. For example, diglycidyl ethers ofbisphenol A can be used, in addition to compounds based on aromaticamine, triazine, and cycloaliphatic backbones. Another example includesthe well-known Novolac polymers. Non-limiting examples of siliconeelastomers suitable for use according to the invention include thoseformed from precursors including the chlorosilanes such asmethylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.

Silicone polymers are preferred, for example, the silicone elastomerpolydimethylsiloxane. Non-limiting examples of PDMS polymers includethose sold under the trademark Sylgard by Dow Chemical Co., Midland,Mich., and particularly Sylgard 182, Sylgard 184, and Sylgard 186.Silicone polymers including PDMS have several beneficial propertiessimplifying formation of the microfluidic structures of the invention.For instance, such materials are inexpensive, readily available, and canbe solidified from a prepolymeric liquid via curing with heat. Forexample, PDMSs are typically curable by exposure of the prepolymericliquid to temperatures of about, for example, about 65° C. to about 75°C. for exposure times of, for example, about an hour. Also, siliconepolymers, such as PDMS, can be elastomeric and thus may be useful forforming very small features with relatively high aspect ratios,necessary in certain embodiments of the invention. Flexible (e.g.,elastomeric) molds or masters can be advantageous in this regard.

The present invention provides improved methods of bonding PDMS toincompatible media. Normal methods of bonding various materials(plastic, metals, etc) directly to materials such as PDMS, silicone,Teflon, and PEEK using traditional bonding practices (adhesives,epoxies, etc) do not work well due to the poor adhesion of the bondingagent to materials such as PDMS. Normal surface preparation bycommercially available surface activators has not worked well inmicrofluidic device manufacturing. This problem is eliminated bytreating the PDMS surface to be bonded with high intensity oxygen or airplasma. The process converts the top layer of PDMS to glass which bondsextremely well with normal adhesives. Tests using this method to bondexternal fluid lines to PDMS using a UV-cure adhesive (Loctite 352, 363,and others) resulted in a bond that is stronger than the PDMS substrate,resulting in fracture of the PDMS prior to failure of the bond. Thepresent method combines high radiant flux, wavelength selection, andcure exposure time to significantly enhance the bond strength of theadhesive.

One advantage of forming structures such as microfluidic structures ofthe invention from silicone polymers, such as PDMS, is the ability ofsuch polymers to be oxidized, for example by exposure to anoxygen-containing plasma such as an air plasma, so that the oxidizedstructures contain, at their surface, chemical groups capable ofcross-linking to other oxidized silicone polymer surfaces or to theoxidized surfaces of a variety of other polymeric and non-polymericmaterials. Thus components can be formed and then oxidized andessentially irreversibly sealed to other silicone polymer surfaces, orto the surfaces of other substrates reactive with the oxidized siliconepolymer surfaces, without the need for separate adhesives or othersealing means. In most cases, sealing can be completed simply bycontacting an oxidized silicone surface to another surface without theneed to apply auxiliary pressure to form the seal. That is, thepre-oxidized silicone surface acts as a contact adhesive againstsuitable mating surfaces. Specifically, in addition to beingirreversibly sealable to itself, oxidized silicone such as oxidized PDMScan also be sealed irreversibly to a range of oxidized materials otherthan itself including, for example, glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, andepoxy polymers, which have been oxidized in a similar fashion to thePDMS surface (for example, via exposure to an oxygen-containing plasma).Methods useful in the context of the present invention, as well asoverall molding techniques, are described in the art, for example, in anarticle entitled “Rapid Prototyping of Microfluidic Systems andPolydimethylsiloxane,” Anal. Chem., 70:474-480, 1998 (Duffy et al.),incorporated herein by reference.

Another advantage to forming microfluidic structures of the invention(or interior, fluid contacting surfaces) from oxidized silicone polymersis that these surfaces can be much more hydrophilic than the surfaces oftypical elastomeric polymers (where a hydrophilic interior surface isdesired). Such hydrophilic channel surfaces can thus be more easilyfilled and wetted with aqueous solutions than can structures comprisedof typical, unoxidized elastomeric polymers or other hydrophobicmaterials.

In one embodiment, a bottom wall is formed of a material different fromone or more side walls or a top wall, or other components. For example,the interior surface of a bottom wall can comprise the surface of asilicon wafer or microchip, or other substrate. Other components can, asdescribed above, be sealed to such alternative substrates. Where it isdesired to seal a component comprising a silicone polymer (e.g. PDMS) toa substrate (bottom wall) of different material, the substrate may beselected from the group of materials to which oxidized silicone polymeris able to irreversibly seal (e.g., glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, epoxy polymers, andglassy carbon surfaces which have been oxidized). Alternatively, othersealing techniques can be used, as would be apparent to those ofordinary skill in the art, including, but not limited to, the use ofseparate adhesives, thermal bonding, solvent bonding, ultrasonicwelding, etc.

Channels

The microfluidic substrates of the present invention include channelsthat form the boundary for a fluid. A “channel,” as used herein, means afeature on or in a substrate that at least partially directs the flow ofa fluid. In some cases, the channel may be formed, at least in part, bya single component, e.g., an etched substrate or molded unit. Thechannel can have any cross-sectional shape, for example, circular, oval,triangular, irregular, square or rectangular (having any aspect ratio),or the like, and can be covered or uncovered (i.e., open to the externalenvironment surrounding the channel). In embodiments where the channelis completely covered, at least one portion of the channel can have across-section that is completely enclosed, and/or the entire channel maybe completely enclosed along its entire length with the exception of itsinlet and outlet.

An open channel generally will include characteristics that facilitatecontrol over fluid transport, e.g., structural characteristics (anelongated indentation) and/or physical or chemical characteristics(hydrophobicity vs. hydrophilicity) and/or other characteristics thatcan exert a force (e.g., a containing force) on a fluid. The fluidwithin the channel may partially or completely fill the channel. In somecases the fluid may be held or confined within the channel or a portionof the channel in some fashion, for example, using surface tension(e.g., such that the fluid is held within the channel within a meniscus,such as a concave or convex meniscus). In an article or substrate, some(or all) of the channels may be of a particular size or less, forexample, having a largest dimension perpendicular to fluid flow of lessthan about 5 mm, less than about 2 mm, less than about 1 mm, less thanabout 500 microns, less than about 200 microns, less than about 100microns, less than about 60, less than about 50 microns, less than about40 microns, less than about 30 microns, less than about 25 microns, lessthan about 10 microns, less than about 3 microns, less than about 1micron, less than about 300 nm, less than about 100 nm, less than about30 nm, or less than about 10 nm or less in some cases. Of course, insome cases, larger channels, tubes, etc. can be used to store fluids inbulk and/or deliver a fluid to the channel. In one embodiment, thechannel is a capillary.

The dimensions of the channel may be chosen such that fluid is able tofreely flow through the channel, for example, if the fluid containscells. The dimensions of the channel may also be chosen, for example, toallow a certain volumetric or linear flow rate of fluid in the channel.Of course, the number of channels and the shape of the channels can bevaried by any method known to those of ordinary skill in the art. Insome cases, more than one channel or capillary may be used. For example,two or more channels may be used, where they are positioned inside eachother, positioned adjacent to each other, etc.

For particles (e.g., cells) or molecules that are in droplets (i.e.,deposited by the inlet module) within the flow of the main channel, thechannels of the device are preferably square, with a diameter betweenabout 2 microns and 1 mm. This geometry facilitates an orderly flow ofdroplets in the channels. Similarly, the volume of the detection modulein an analysis device is typically in the range of between about 0.1picoliters and 500 nanoliters.

A “main channel” is a channel of the device of the invention whichpermits the flow of molecules, cells, small molecules or particles pasta coalescence module for coalescing one or more droplets, a detectionmodule for detection (identification) or measurement of a droplet and asorting module, if present, for sorting a droplet based on the detectionin the detection module. The main channel is typically in fluidcommunication with the coalescence, detection and/or sorting modules, aswell as, an inlet channel of the inlet module. The main channel is alsotypically in fluid communication with an outlet module and optionallywith branch channels, each of which may have a collection module orwaste module. These channels permit the flow of molecules, cells, smallmolecules or particles out of the main channel. An “inlet channel”permits the flow of molecules, cells, small molecules or particles intothe main channel. One or more inlet channels communicate with one ormore means for introducing a sample into the device of the presentinvention. The inlet channel communicates with the main channel at aninlet module.

The microfluidic substrate can also comprise one or more fluid channelsto inject or remove fluid in between droplets in a droplet stream forthe purpose of changing the spacing between droplets.

The channels of the device of the present invention can be of anygeometry as described. However, the channels of the device can comprisea specific geometry such that the contents of the channel aremanipulated, e.g., sorted, mixed, prevent clogging, etc.

A microfluidic substrate can also include a specific geometry designedin such a manner as to prevent the aggregation of biological/chemicalmaterial and keep the biological/chemical material separated from eachother prior to encapsulation in droplets. The geometry of channeldimension can be changed to disturb the aggregates and break them apartby various methods, that can include, but is not limited to, geometricpinching (to force cells through a (or a series of) narrow region(s),whose dimension is smaller or comparable to the dimension of a singlecell) or a barricade (place a series of barricades on the way of themoving cells to disturb the movement and break up the aggregates ofcells).

To prevent material (e.g., cells and other particles or molecules) fromadhering to the sides of the channels, the channels (and coverslip, ifused) may have a coating which minimizes adhesion. Such a coating may beintrinsic to the material from which the device is manufactured, or itmay be applied after the structural aspects of the channels have beenmicrofabricated. “TEFLON” is an example of a coating that has suitablesurface properties. The surface of the channels of the microfluidicdevice can be coated with any anti-wetting or blocking agent for thedispersed phase. The channel can be coated with any protein to preventadhesion of the biological/chemical sample. For example, in oneembodiment the channels are coated with BSA, PEG-silane and/orfluorosilane. For example, 5 mg/ml BSA is sufficient to preventattachment and prevent clogging. In another embodiment, the channels canbe coated with a cyclized transparent optical polymer obtained bycopolymerization of perfluoro (alkenyl vinyl ethers), such as the typesold by Asahi Glass Co. under the trademark Cytop. In such anembodiment, the coating is applied from a 0.1-0.5 wt % solution of CytopCTL-809M in CT-Solv 180. This solution can be injected into the channelsof a microfluidic device via a plastic syringe. The device can then beheated to about 90° C. for 2 hours, followed by heating at 200° C. foran additional 2 hours. In another embodiment, the channels can be coatedwith a hydrophobic coating of the type sold by PPG Industries, Inc.under the trademark Aquapel (e.g., perfluoroalkylsilane surfacetreatment of plastic and coated plastic substrate surfaces inconjunction with the use of a silica primer layer) and disclosed in U.S.Pat. No. 5,523,162, which patient is hereby incorporated by reference.By fluorinating the surfaces of the channels, the continuous phasepreferentially wets the channels and allows for the stable generationand movement of droplets through the device. The low surface tension ofthe channel walls thereby minimizes the accumulation of channel cloggingparticulates.

The surface of the channels in the microfluidic device can be alsofluorinated to prevent undesired wetting behaviors. For example, amicrofluidic device can be placed in a polycarbonate dessicator with anopen bottle of (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane.The dessicator is evacuated for 5 minutes, and then sealed for 20-40minutes. The dessicator is then backfilled with air and removed. Thisapproach uses a simple diffusion mechanism to enable facile infiltrationof channels of the microfluidic device with the fluorosilane and can bereadily scaled up for simultaneous device fluorination.

Fluids

The microfluidic device of the present invention is capable ofcontrolling the direction and flow of fluids and entities within thedevice. The term “flow” means any movement of liquid or solid through adevice or in a method of the invention, and encompasses withoutlimitation any fluid stream, and any material moving with, within oragainst the stream, whether or not the material is carried by thestream. For example, the movement of molecules, beads, cells or virionsthrough a device or in a method of the invention, e.g. through channelsof a microfluidic chip of the invention, comprises a flow. This is so,according to the invention, whether or not the molecules, beads, cellsor virions are carried by a stream of fluid also comprising a flow, orwhether the molecules, cells or virions are caused to move by some otherdirect or indirect force or motivation, and whether or not the nature ofany motivating force is known or understood. The application of anyforce may be used to provide a flow, including without limitation,pressure, capillary action, electro-osmosis, electrophoresis,dielectrophoresis, optical tweezers, and combinations thereof, withoutregard for any particular theory or mechanism of action, so long asmolecules, cells or virions are directed for detection, measurement orsorting according to the invention. Specific flow forces are describedin further detail herein.

The flow stream in the main channel is typically, but not necessarily,continuous and may be stopped and started, reversed or changed in speed.A liquid that does not contain sample molecules, cells or particles canbe introduced into a sample inlet well or channel and directed throughthe inlet module, e.g., by capillary action, to hydrate and prepare thedevice for use. Likewise, buffer or oil can also be introduced into amain inlet region that communicates directly with the main channel topurge the device (e.g., or “dead” air) and prepare it for use. Ifdesired, the pressure can be adjusted or equalized, for example, byadding buffer or oil to an outlet module.

As used herein, the term “fluid stream” or “fluidic stream” refers tothe flow of a fluid, typically generally in a specific direction. Thefluidic stream may be continuous and/or discontinuous. A “continuous”fluidic stream is a fluidic stream that is produced as a single entity,e.g., if a continuous fluidic stream is produced from a channel, thefluidic stream, after production, appears to be contiguous with thechannel outlet. The continuous fluidic stream is also referred to as acontinuous phase fluid or carrier fluid. The continuous fluidic streammay be laminar, or turbulent in some cases.

Similarly, a “discontinuous” fluidic stream is a fluidic stream that isnot produced as a single entity. The discontinuous fluidic stream isalso referred to as the dispersed phase fluid or sample fluid. Adiscontinuous fluidic stream may have the appearance of individualdroplets, optionally surrounded by a second fluid. A “droplet,” as usedherein, is an isolated portion of a first fluid that completelysurrounded by a second fluid. In some cases, the droplets may bespherical or substantially spherical; however, in other cases, thedroplets may be spherical or substantially spherical; however, in othercases, the droplets may be non-spherical, for example, the droplets mayhave the appearance of “blobs” or other irregular shapes, for instance,depending on the external environment. As used herein, a first entity is“surrounded” by a second entity if a closed loop can be drawn oridealized around the first entity through only the second entity. Thedispersed phase fluid can include a biological/chemical material. Thebiological/chemical material can be tissues, cells, particles, proteins,antibodies, amino acids, nucleotides, small molecules, andpharmaceuticals. The biological/chemical material can include one ormore labels known in the art. The label can be a DNA tag, dyes orquantum dot, or combinations thereof.

Droplets

The term “emulsion” refers to a preparation of one liquid distributed insmall globules (also referred to herein as drops, droplets orNanoReactors) in the body of a second liquid. The first and secondfluids are immiscible with each other. For example, the discontinuousphase can be an aqueous solution and the continuous phase can ahydrophobic fluid such as an oil. This is termed a water in oilemulsion. Alternatively, the emulsion may be a oil in water emulsion. Inthat example, the first liquid, which is dispersed in globules, isreferred to as the discontinuous phase, whereas the second liquid, isreferred to as the continuous phase or the dispersion medium. Thecontinuous phase can be an aqueous solution and the discontinuous phaseis a hydrophobic fluid, such as an oil (e.g., decane, tetradecane, orhexadecane). The droplets or globules of oil in an oil in water emulsionare also referred to herein as “micelles”, whereas globules of water ina water in oil emulsion may be referred to as “reverse micelles”.

The fluidic droplets may each be substantially the same shape and/orsize. The shape and/or size can be determined, for example, by measuringthe average diameter or other characteristic dimension of the droplets.The “average diameter” of a plurality or series of droplets is thearithmetic average of the average diameters of each of the droplets.Those of ordinary skill in the art will be able to determine the averagediameter (or other characteristic dimension) of a plurality or series ofdroplets, for example, using laser light scattering, microscopicexamination, or other known techniques. The diameter of a droplet, in anon-spherical droplet, is the mathematically-defined average diameter ofthe droplet, integrated across the entire surface. The average diameterof a droplet (and/or of a plurality or series of droplets) may be, forexample, less than about 1 mm, less than about 500 micrometers, lessthan about 200 micrometers, less than about 100 micrometers, less thanabout 75 micrometers, less than about 50 micrometers, less than about 25micrometers, less than about 10 micrometers, or less than about 5micrometers in some cases. The average diameter may also be at leastabout 1 micrometer, at least about 2 micrometers, at least about 3micrometers, at least about 5 micrometers, at least about 10micrometers, at least about 15 micrometers, or at least about 20micrometers in certain cases.

As used herein, the term “NanoReactor” and its plural encompass theterms “droplet”, “nanodrop”, “nanodroplet”, “microdrop” or“microdroplet” as defined herein, as well as an integrated system forthe manipulation and probing of droplets, as described in detail herein.Nanoreactors as described herein can be 0.1-1000 μm (e.g., 0.1, 0.2 . .. 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 . . . 1000), or any size withinin this range. Droplets at these dimensions tend to conform to the sizeand shape of the channels, while maintaining their respective volumes.Thus, as droplets move from a wider channel to a narrower channel theybecome longer and thinner, and vice versa.

The microfluidic substrate of this invention most preferably generateround, monodisperse droplets. The droplets can have a diameter that issmaller than the diameter of the microchannel; i.e., preferably 15 to100 μm when cells are used; or 10 to 75 μm when reagents or otherchemical or biological agents are used; or 100 to 1000 μm when dropletsare used for sequencing reactions such that droplets will be removed anddispensed into other collection apparatuses, such as microtiter platesor utilized in sequencing devices. Monodisperse droplets areparticularly preferably, e.g., in high throughput devices and otherembodiments where it is desirable to generate droplets at high frequencyand of high uniformity.

The droplet forming liquid is typically an aqueous buffer solution, suchas ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for exampleby column chromatography), 10 mM Tris HCl and 1 mM EDTA (TE) buffer,phosphate buffer saline (PBS) or acetate buffer. Any liquid or bufferthat is physiologically compatible with the population of molecules,cells or particles to be analyzed and/or sorted can be used. The fluidpassing through the main channel and in which the droplets are formed isone that is immiscible with the droplet forming fluid. The fluid passingthrough the main channel can be a non-polar solvent, decane (e g.,tetradecane or hexadecane), fluorocarbon oil, silicone oil or anotheroil (for example, mineral oil).

The dispersed phase fluid may also contain biological/chemical material(e.g., molecules, cells or other particles) for combination, analysisand/or sorting in the device. The droplets of the dispersed phase fluidcan contain more than one particle or can contain no more than oneparticle. For example, where the biological material comprises cells,each droplet preferably contains, on average, no more than one cell.However, in some embodiments, each droplet may contain, on average, atleast 1000 cells. The droplets can be detected and/or sorted accordingto their contents.

The concentration (i.e., number) of molecules, cells or particles in adroplet can influence sorting efficiently and therefore is preferablyoptimized. In particular, the sample concentration should be diluteenough that most of the droplets contain no more than a single molecule,cell or particle, with only a small statistical chance that a dropletwill contain two or more molecules, cells or particles. This is toensure that for the large majority of measurements, the level ofreporter measured in each droplet as it passes through the detectionmodule corresponds to a single molecule, cell or particle and not to twoor more molecules, cells or particles.

The parameters which govern this relationship are the volume of thedroplets and the concentration of molecules, cells or particles in thesample solution. The probability that a droplet will contain two or moremolecules, cells or particles (P_(≤2)) can be expressed as

P _(≤2)=1−{1+[cell]×V}×e ^(−[cell]×V)

where “[cell]” is the concentration of molecules, cells or particles inunits of number of molecules, cells or particles per cubic micron (μm³),and V is the volume of the droplet in units of μm^(3.)

It will be appreciated that P_(≤2) can be minimized by decreasing theconcentration of molecules, cells or particles in the sample solution.However, decreasing the concentration of molecules, cells or particlesin the sample solution also results in an increased volume of solutionprocessed through the device and can result in longer run times.Accordingly, it is desirable to minimize to presence of multiplemolecules, cells or particles in the droplets (thereby increasing theaccuracy of the sorting) and to reduce the volume of sample, therebypermitting a sorted sample in a reasonable time in a reasonable volumecontaining an acceptable concentration of molecules, cells or particles.

The maximum tolerable P_(≤2) depends on the desired “purity” of thesorted sample. The “purity” in this case refers to the fraction ofsorted molecules, cells or particles that posses a desiredcharacteristic (e.g., display a particular antigen, are in a specifiedsize range or are a particular type of molecule, cell or particle). Thepurity of the sorted sample is inversely proportional to P_(≤2). Forexample, in applications where high purity is not needed or desired arelatively high P_(<2) (e.g., P_(<2)=0.2) may be acceptable. For mostapplications, maintaining P_(<2) at or below about 0.1, preferably at orbelow about 0.01, provides satisfactory results.

The fluids used to generate droplets in microfluidic devices aretypically immiscible liquids such as oil and water. These two materialsgenerally have very different dielectric constants associated with them.These differences can be exploited to determine droplet rate and sizefor every drop passing through a small section of a microfluidic device.One method to directly monitor this variation in the dielectric constantmeasures the change in capacitance over time between a pair of closelyspaced electrodes. This change in capacitance can be detected by thechange in current measured in these electrodes:

$i = {V\mspace{14mu} \frac{dC}{dt}}$

Where i is the current, V is the voltage applied across the electrodes,and dC/dt is the change in capacitance with time. Alternatively, thecapacitance can be measured directly if a time varying voltage isapplied to these same electrodes: i=CdV/dt Where C is the measuredcapacitance, and dV/dt is the change in voltage with time. As a firstapproximation, the electrode pair can be determined as a parallel platecapacitor:

$C = {ɛ_{0}k\mspace{14mu} \frac{A}{d}}$

Where

₀ is the permittivity of free space, k is the effective dielectricconstant (this changes every time a droplet passes through), A is thearea of the capacitor and d is the electrode separation. The currentmeasured in the device is then plotted as a function of time.

The fluidic droplets may contain additional entities, for example, otherchemical, biochemical, or biological entities (e.g., dissolved orsuspended in the fluid), cells, particles, gases, molecules, or thelike. In some cases, the droplets may each be substantially the sameshape or size, as discussed above. In certain instances, the inventionprovides for the production of droplets consisting essentially of asubstantially uniform number of entities of a species therein (i.e.,molecules, cells, particles, etc.). For example, about 90%, about 93%,about 95%, about 97%, about 98%, or about 99%, or more of a plurality orseries of droplets may each contain the same number of entities of aparticular species. For instance, a substantial number of fluidicdroplets produced, e.g., as described above, may each contain 1 entity,2 entities, 3 entities, 4 entities, 5 entities, 7 entities, 10 entities,15 entities, 20 entities, 25 entities, 30 entities, 40 entities, 50entities, 60 entities, 70 entities, 80 entities, 90 entities, 100entities, etc., where the entities are molecules or macromolecules,cells, particles, etc. In some cases, the droplets may eachindependently contain a range of entities, for example, less than 20entities, less than 15 entities, less than 10 entities, less than 7entities, less than 5 entities, or less than 3 entities in some cases.In some embodiments, a droplet may contain 100,000,000 entities. Inother embodiments, a droplet may contain 1,000,000 entities.

In a liquid containing droplets of fluid, some of which contain aspecies of interest and some of which do not contain the species ofinterest, the droplets of fluid may be screened or sorted for thosedroplets of fluid containing the species as further described below(e.g., using fluorescence or other techniques such as those describedabove), and in some cases, the droplets may be screened or sorted forthose droplets of fluid containing a particular number or range ofentities of the species of interest, e.g., as previously described.Thus, in some cases, a plurality or series of fluidic droplets, some ofwhich contain the species and some of which do not, may be enriched (ordepleted) in the ratio of droplets that do contain the species, forexample, by a factor of at least about 2, at least about 3, at leastabout 5, at least about 10, at least about 15, at least about 20, atleast about 50, at least about 100, at least about 125, at least about150, at least about 200, at least about 250, at least about 500, atleast about 750, at least about 1000, at least about 2000, or at leastabout 5000 or more in some cases. In other cases, the enrichment (ordepletion) may be in a ratio of at least about 10⁴, at least about 10⁵,at least about 10⁶, at least about 10⁷, at least about 10⁸, at leastabout 10⁹, at least about 10¹⁰, at least about 10¹¹, at least about10¹²; at least about 10¹³, at least about 10¹⁴, at least about 10¹⁵, ormore. For example, a fluidic droplet containing a particular species maybe selected from a library of fluidic droplets containing variousspecies, where the library may have about 100, about 10³, about 10⁴,about 10⁵, about 10⁶, about 10⁷, about 10⁸, about 10⁹, about 10¹⁰, about10¹¹, about 10¹², about 10¹³, about 10¹⁴, about 10¹⁵, or more items, forexample, a DNA library, an RNA library, a protein library, acombinatorial chemistry library, etc. In certain embodiments, thedroplets carrying the species may then be fused, reacted, or otherwiseused or processed, etc., as further described below, for example, toinitiate or determine a reaction.

Droplets of a sample fluid can be formed within the inlet module on themicrofluidic device or droplets (or droplet libraries) can be formedbefore the sample fluid is introduced to the microfluidic device (“offchip” droplet formation). To permit effective interdigitation,coalescence and detection, the droplets comprising each sample to beanalyzed must be monodisperse. As described in more detail herein, inmany applications, different samples to be analyzed are contained withindroplets of different sizes. Droplet size must be highly controlled toensure that droplets containing the correct contents for analysis andcoalesced properly. As such, the present invention provides devices andmethods for forming droplets and droplet libraries.

Devices and Methods for Forming Sample Droplets on a MicrofluidicSubstrate

The present invention provides compositions and methods for formingsample droplet emulsions on a microfluidic substrate. The presentinvention also provides embedded microfluidic nozzles. In order tocreate a monodisperse emulsion directly from a library well, thisinvention would form a nozzle directly into the fitting used to connectthe storage well/reservoir (e.g. syringe) to a syringe tip (e.g.capillary tubing), as shown in FIGS. 2-6. FIG. 2, Panels A and B, showdual and single oil versions of the nozzle concept using a small ferrulefor the nozzle section. FIG. 2, Panels C and D, show the same nozzlesmade directly out of small bore tubing (the “nozzle” runs the entirelength of the tubing). Both designs can form droplets identically,although the pressure drop will be higher for the tube based nozzle(bottom). FIG. 3 shows the expansion of the nozzle ferrule concept shownin FIGS. 2A and 2B. The tube based nozzles (FIGS. 2C, 2D) functionidentically to this, except the “nozzle” runs the entire length of thetube instead of having a short transition. The ability to form dropletsis identical in both cases. FIG. 4 shows the expansion of the nozzlesection contained in the ferrule. The tee design in FIG. 2D has beenbuilt and tested, with a cross-section cut of this design shown in FIG.5. FIG. 5A shows the operation of the nozzle in Aspiration Mode and FIG.5B shows the operation of the nozzle in Injection Mode. The dropletsformed are approximately 45 um in diameter, and were formed from PCR mix(210 ul/hr) and SpectraSyn-10 (600 ul/hr). Other tests have beendemonstrated with Spectrasyn-2 and PCR mix. The droplets are travelingin 300 um wide×260 um deep channels. The nozzle tube used was 100 um indiameter, and the fluids used were PCR Mix and Spectrasyn-10 withsurfactant.

Since the flow is three dimensional, under this design surface wettingeffects are minimized. The nozzle can be made from one or two oil linesproviding constant flow of oil into the nozzle, a connection to thecapillary tubing, and a connection to the storage well/reservoir (e.g.syringe). The high resolution part of the nozzle can be made out of asmall bore tubing or a small, simple part molded or stamped from anappropriate material (Teflon®, plastic, metal, etc). If necessary, thenozzle itself could be formed into the tip of the ferrule using postmold processing such as laser ablation or drilling.

This nozzle design eliminates the surface wetting issues surrounding thequasi-2D flow associated with typical microfluidic nozzles made usingsoft lithography or other standard microfluidic chip manufacturingtechniques. This is because the nozzle design is fully 3-dimensional,resulting is a complete isolation of the nozzle section from thecontinuous aqueous phase. This same design can also be used forgeneration of emulsions required for immediate use, where the aqueousline would be attached directly to a syringe and the outlet of thenozzle would be used to transport the emulsion to the point’ of use(e.g. into a microfluidic PCR chip, delay line, etc).

In another embodiment, the present invention provides compositions andmethods to directly emulsify library elements from standard librarystorage geometries (e.g. 96 well plates, etc). In order to create amonodisperse emulsion from fluids contained in a library well plate,this invention would include microfluidic based nozzles manufacturedsimultaneously with an appropriately designed fluidic interconnect orwell. FIGS. 6 and 7 present two possible approaches to interface withthe nozzle.

FIG. 6 shows a reservoir based sample emulsification. In FIG. 6, thewell is initially filled with a fluid with lower density than the sampleto be introduced. The operation of this device would be very similar tothe device described above, with the exception that the sample would beintroduced into a port instead of being directly aspirated from a samplewell. This could either be emulsification oil obtained directly from thenozzle, or a different material that is loaded or flowing into the wellautomatically. The oil lines would begin flowing at their prescribedrates (FIG. 6e ), while the collection or waste port would beginwithdrawing at a rate corresponding to the total oil flow plus thedesired sample flow. Once the flow has been established, the samplewould be introduced into the port either manually (e.g. a pipette) orwith a robotic sample handling system. The sample volume permitted wouldbe dependent on the port volume. Since the sample is more dense than thefluid in the well, it would settle into the bottom of the well and betransported to the nozzle (FIGS. 6a-6d ). During this time, either thewaste (used during startup only if transients cause problems) or thecollection port would be withdrawing emulsified sample and stored. Whenthe sample is completely emulsified the next sample would be introducedand the process repeated. If washing steps are required between runs,the washing fluids would be withdrawn into the waste line. If thepressure drop across the nozzle would cause cavitation on collectionthen an optional pressurization of the input well can be utilized.

FIG. 7 shows sample introduction when the sample is less dense than thefluid in the sample port. FIG. 7 depicts an alternative scheme thatcould be used to introduce samples that are less dense than the oil usedto emulsify the sample. As with the concept in FIG. 1, the process ofintroducing the sample into the port could be run either manually orwith a robotic sampling system. In this concept, the sample port couldbe filled with the emulsification oil through backflow from the nozzleprior to introduction of the sample (FIG. 7a ). If this oil is notappropriate, the port can be filled from the top with a differentimmiscible fluid that might have more desirable properties than theemulsification oil (e.g. better wetting, less surfactant, etc). Thissecond immiscible fluid could be introduced during startup and flowcontinuously into the port when the sample tip is not inserted. Keepingthe sample port filled with fluid will prevent air entrainment duringstartup and should improve transient performance.

If the sample tip is connected to a pump capable of driving the sampleinto the device, it could be started up as the tip is inserted into thedevice (7 b-c). When used with this sort of sample introduction, thedevice could be run identically to the “normal” operation of ourdevices, including having the “transport to waste” line (7 e) notconnected to a pump. If the sample tip loading pump is not capable ofaccurately forcing the flow (i.e. not connected to a suitable pump), theback end of the tip could be connected to a valve that would open toeither atmospheric pressure (or possibly a pressurized gas supply) whenthe tip is fully inserted into the port. In order to prevent airentrainment into the sample tip and device, this connection could bemade through a reservoir filled with the desired immiscible liquid. Ineither case, the device would run identically to the one described aboveand shown in FIG. 6. FIG. 7 also shows another possible configuration ofthe aspiration probe assembly used for the device in FIG. 6.

Methods for Forming Sample Droplet Emulsions Prior to Injection on aMicrofluidic Substrate

The present invention also provides compositions and methods forcreating emulsion of the sample fluid (e.g. droplets) prior to theintroduction of the sample fluid into the microfluidic devices of thepresent invention. More specifically, the methods are directed to thecreating sample droplet emulsions “off chip”, for example in a syringe.In order to create a monodisperse emulsion directly from a library well,a nozzle is formed directly into the fitting used to connect thecollection syringe to a syringe tip (e.g. capillary tubing), as shown inFIG. 8. The nozzle can be made from one or two oil lines providingconstant flow of oil into the nozzle, a connection to the capillarytubing, and a connection to the collection syringe. Aspiration of thesample can be accomplished by running the collection syringe inwithdrawal mode at a flow rate (Q3) above the flow rate of the two oilsyringes (Step 1 in FIG. 8). The difference in flow would correspond tothe flow rate aspirated from the sample well. When the appropriatevolume of sample has been loaded into the capillary tubing, thecapillary tubing would be removed from the sample well, an air bubble,and possibly a cleaning solution would be aspirated (Step 2 in FIG. 8).When almost all of the sample has been emulsified, the collectionsyringe withdrawal rate would either be reduced below the oil flowrates, stopped, or set to infuse at some nominal rate (Step 3 in FIG.8). The remaining sample, air, cleaning solution, etc, left in thecapillary would be flushed back out into a cleaning well and the outsideof the capillary would be cleaned at the “wash station.” When thecapillary is completely clean, the process would repeat for the nextlibrary element.

The nozzle can be formed through using small bore tubing (glass,Teflon®, PEEK tubing or capillaries) or micro-fabrication or moldingprocesses such as PDMS soft lithography, glass etching, hot embossing,or similar high resolution fabrication technology.

The present apparatus can be readily adapted for clinical applicationsor work where cross contamination must be eliminated, since the regionfrom the nozzle to the syringe are isolated from the sample stream(e.g., the oil wets these surfaces and keeps the sample from directlycontacting aqueous sample). The aspiration tip can be designed as adisposable item (like a robotic sampler aspiration tips) andautomatically replaced between samples.

Multiple nozzle/syringe pairs can be operated in parallel, thusincreasing throughput. This allows simultaneous sampling of multiplewells/samples during a single process step. Each sample can be collectedinto a separate syringe.

Other methods for forming sample droplet emulsions and emulsionlibraries “off chip” are described in Example 1.

Methods for Minimizing Sample Volume Loss

As described herein, a significant problem when working with very smallamounts of reagents comes from the losses associated with dead volumesfound in the storage containers and transport lines. As an example, if50 microliters of a material is injected into a 254 micron internaldiameter capillary tube, a 75 cm long tube would consume about 38microliters of the material (˜75%). To address this problem, the presentinvention provides compositions and methods which eliminates theproblems associated with dead volume and reagent waste when working withextremely small volumes of reagents.

In one embodiment, the primary reagents (sample) is combined with asecond, immiscible phase in the storage container (e.g. a syringe orother reservoir). This second phase is used to push the entire amount ofthe first phase into the system with no significant losses. Morespecifically, when two immiscible fluids are combined in a reservoir,the two fluids will tend to separate into layers as long as thedensities of the materials are different. If the fluid of interest(e.g., sample fluid) is closest to the exit of the reservoir, it will bethe first to leave when the reservoir is emptied (the exit can be oneither the top or bottom, depending on the density difference). Once thereagent has been pumped out of the reservoir, the second phase willfollow. This second phase will then push the first phase completelythrough the system without any sample fluid loss. As an example of this,oil and water (the reagent) would be combined in a syringe. If the oilis denser than the water, the syringe would be oriented with its exitface up, if the oil were less dense, then the syringe would be facedown. The oil would be chosen such that the materials of interest in thereagent are not soluble in the oil phase. FIG. 9 is one example of thisapproach when a syringe is used as the reservoir and the second phase isdenser than the reagent phase. If the reagent were more dense, then thesyringe orientation would be reversed (i.e. the exit would be facingdownward in the figure). Specifically, FIG. 9 shows a two phase systemwhere the reagent is injected on top of the second, immiscible phase:(A) During injection, prior to transition from first phase to secondphase, (B) second phase just entering the transfer line, (C) secondphase has completely filled the transfer line and pushed the entirevolume of reagent through the system.

Alternatively, a sample solution is sandwiched between two immiscibleliquids, wherein one liquid has a density greater than the sampledensity, and the second liquid has a density less than the sampledensity. Referring to FIG. 10, the sample (density 1.0) can be layeredbetween perflourocarbon oil (density 1.8) and mineral oil (density0.914). Ideally, when the device is being used to analyze biologicalreactions the immiscible solutions do not inhibit the reactions of thesample, nor are any test molecules in the sample or the sample itselfsoluble in either immiscible fluid. The less dense fluid (in FIG. 10,the mineral oil) can be used to ‘prime the pump’ and remove any air ordead-space that occurs during normal injection. The sample then rises tothe injection point after the mineral oil. It is further contemplatedthat the methods disclosed herein would also work for gases. The gasesand/or liquids can be miscible, but of different densities such thatthey are layered on top each other in a manner that prevents theirmixing.

Solid or Semi-Solid Phase Droplets

The present invention also provides solid phase particles and methodsfor the forming solid phase particles on a microfluidic device fordownstream analysis. The solid phase particles can be used for variousbiological or chemical analysis (e.g., DNA or protein analyses).

For DNA analysis, post amplification encapsulation of amplicons occurswithin a gel or polymer matrix prior to breaking of the dropletemulsion. Amplification reactions within droplets using one of severalamplification type methods (described in further detail herein),including, but not limited to; PCR, Rolling Circle Amplification (RCA),Nucleic Acid Sequence Based Amplification (NASBA), ligase chainreaction, etc. followed by encapsulation/solidification of the amplifiedreaction within the droplets by either polymerizing the droplets usingchemical or physical means.

A physical means might be termed ‘gelling’ whereby one incorporates lowtemperature agarose within the droplet during formulation and keepingthe droplet above the solidification temperature until one desires thedroplet to solidify.

A chemical means might be termed ‘polymerization’ whereby one combines(if needed) the droplet with a polymerizing solution and thenpolymerizing the droplet using either a polymerization initiator (forexample free radicals) or a means such as UV light. Some other means ofgelling or polymerization include matragel, polyacrylamide, mixedpolysaccharides, etc. Some example initiators can be temperature, UVirradiation, etc.

In a further example, one of the DNA primers used for amplification canbe attached to one of the molecules that will form the polymerizedmatrix. Attachment can be through a direct chemical attachment, orthrough a secondary attachment such as biotin-streptavidin attachment.In this example, the DNA will become physically attached to the formedsolid-phase that occurs after solidification of the droplet. Using oneof several gelling or polymerizing methods it should be possible tofurther manipulate these droplets.

One could also either exchange or wash away unincorporated nucleotides,primers and exchange buffers so as to remove the initial amplificationbuffers, polymerases, etc. In an example of further droplet manipulationwherein one of the strands is polymerized from an attached DNAamplification primer, one could treat the polymerized or gelled dropletwith a base solution to disassociate the two DNA strands and elute, fromthe gelled or polymerized droplet, the unattached strand.

For protein analysis, proteins can be either trapped within, or attachedto the gel or polymer matrix. If attached, it can be through a covalentlinkage or through an affinity tag, for example his6 or avi-tag. Theproteins can be added to droplets containing gel or polymer reagent, orthey can be formulated along with the gel or polymerization reagent.Variations that include both are also possible. The protein can be addedto the droplets. Additionally, it is possible to add DNA to the dropletand allow in vitro transcription/translation to synthesize the protein.

The droplets can be kept in liquid form on the microfluidic device andeither gelled or polymerized upon removal, or can be gelled orpolymerized within the droplets anywhere on the device after thedroplets have been formed.

In an example, multiple plasmids are formulated into a droplet alongwith an in vitro transcription/translation reaction. Genes, encoded bythe plasmids, are translated and transcribed to protein molecules. Theprotein molecules attach to the polymer via an avi-tag, the droplets areallowed to gel and the plasmid molecules become ‘fixed’ or ‘trapped’within the gel. The gelled droplets are collected, the emulsion isbroken and the solidified droplets collected and washed. As an exampleapplication, DNA is amplified within a droplet wherein one primer isphysically attached to a polymer monomer. The droplet is then combinedwith a droplet containing the enzymes DNA polymerase, luciferase andsulfurylase. The merged droplets are allowed to gel or polymerize, theyare collected, and if needed, washed. These washed gelled droplets canthen used for a DNA sequencing reaction.

Surfactants

The fluids used in the invention may contain one or more additives, suchas agents which reduce surface tensions (surfactants). Surfactants caninclude Tween, Span, fluorosurfactants, and other agents that aresoluble in oil relative to water. In some applications, performance isimproved by adding a second surfactant to the aqueous phase. Surfactantscan aid in controlling or optimizing droplet size, flow and uniformity,for example by reducing the shear force needed to extrude or injectdroplets into an intersecting channel. This can affect droplet volumeand periodicity, or the rate or frequency at which droplets break offinto an intersecting channel. Furthermore, the surfactant can serve tostabilize aqueous emulsions in fluorinated oils from coalescing.

The droplets may be coated with a surfactant. Preferred surfactants thatmay be added to the continuous phase fluid include, but are not limitedto, surfactants such as sorbitan-based carboxylic acid esters (e.g., the“Span” surfactants, Fluka Chemika), including sorbitan monolaurate (Span20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60)and sorbitan monooleate (Span 80), and perfluorinated polyethers (e.g.,DuPont Krytox 157 FSL, FSM, and/or FSH). Other non-limiting examples ofnon-ionic surfactants which may be used include polyoxyethylenatedalkylphenols (for example, nonyl-, p-dodecyl-, and dinonylphenols),polyoxyethylenated straight chain alcohols, polyoxyethylenatedpolyoxypropylene glycols, polyoxyethylenated mercaptans, long chaircarboxylic acid esters (for example, glyceryl and polyglycerl esters ofnatural fatty acids, propylene glycol, sorbitol, polyoxyethylenatedsorbitol esters, polyoxyethylene glycol esters, etc.) and alkanolamines(e.g., diethanolamine-fatty acid condensates and isopropanolamine-fattyacid condensates). In addition, ionic surfactants such as sodium dodecylsulfate (SDS) may also be used. However, such surfactants are generallyless preferably for many embodiments of the invention. For instance, inthose embodiments where aqueous droplets are used as nanoreactors forchemical reactions (including biochemical reactions) or are used toanalyze and/or sort biomaterials, a water soluble surfactant such as SDSmay denature or inactivate the contents of the droplet.

The carrier fluid can be an oil (e.g., decane, tetradecane orhexadecane) or fluorocarbon oil that contains a surfactant (e.g., anon-ionic surfactant such as a Span surfactant) as an additive(preferably between about 0.2 and 5% by volume, more preferably about2%). A user can preferably cause the carrier fluid to flow throughchannels of the microfluidic device so that the surfactant in thecarrier fluid coats the channel walls.

In one embodiment, the fluorosurfactant can be prepared by reacting theperflourinated polyether DuPont Krytox 157 FSL, FSM, or FSH with aqueousammonium hydroxide in a volatile fluorinated solvent. The solvent andresidual water and ammonia can be removed with a rotary evaporator. Thesurfactant can then be dissolved (e.g., 2.5 wt %) in a fluorinated oil(e.g., Flourinert (3M)), which then serves as the continuous phase ofthe emulsion.

Driving Forces

The invention can use pressure drive flow control, e.g., utilizingvalves and pumps, to manipulate the flow of cells, particles, molecules,enzymes or reagents in one or more directions and/or into one or morechannels of a microfluidic device. However, other methods may also beused, alone or in combination with pumps and valves, such aselectro-osmotic flow control, electrophoresis and dielectrophoresis(Fulwyer, Science 156, 910 (1974); Li and Harrison, Analytical Chemistry69, 1564 (1997); Fiedler, et al. Analytical Chemistry 70, 1909-1915(1998); U.S. Pat. No. 5,656,155). Application of these techniquesaccording to the invention provides more rapid and accurate devices andmethods for analysis or sorting, for example, because the sorting occursat or in a sorting module that can be placed at or immediately after adetection module. This provides a shorter distance for molecules orcells to travel, they can move more rapidly and with less turbulence,and can more readily be moved, examined, and sorted in single file,i.e., one at a time.

Positive displacement pressure driven flow is a preferred way ofcontrolling fluid flow and dielectrophoresis is a preferred way ofmanipulating droplets within that flow.

The pressure at the inlet module can also be regulated by adjusting thepressure on the main and sample inlet channels, for example, withpressurized syringes feeding into those inlet channels. By controllingthe pressure difference between the oil and water sources at the inletmodule, the size and periodicity of the droplets generated may beregulated. Alternatively, a valve may be placed at or coincident toeither the inlet module or the sample inlet channel connected thereto tocontrol the flow of solution into the inlet module, thereby controllingthe size and periodicity of the droplets. Periodicity and droplet volumemay also depend on channel diameter, the viscosity of the fluids, andshear pressure.

Without being bound by any theory, electro-osmosis is believed toproduce motion in a stream containing ions e.g. a liquid such as abuffer, by application of a voltage differential or charge gradientbetween two or more electrodes. Neutral (uncharged) molecules or cellscan be carried by the stream. Electro-osmosis is particularly suitablefor rapidly changing the course, direction or speed of flow.Electrophoresis is believed to produce movement of charged objects in afluid toward one or more electrodes of opposite charge, and away fromone on or more electrodes of like charge. Where an aqueous phase iscombined with an oil phase, aqueous droplets are encapsulated orseparated from each other by oil. Typically, the oil phase is not anelectrical conductor and may insulate the droplets from theelectro-osmotic field. In this example, electro-osmosis may be used todrive the flow of droplets if the oil is modified to carry or react toan electrical field, or if the oil is substituted for another phase thatis immiscible in water but which does not insulate the water phase fromelectrical fields.

Dielectrophoresis is believed to produce movement of dielectric objects,which have no net charge, but have regions that are positively ornegatively charged in relation to each other. Alternating,non-homogeneous electric fields in the presence of droplets and/orparticles, such as cells or molecules, cause the droplets and/orparticles to become electrically polarized and thus to experiencedielectrophoretic forces. Depending on the dielectric polarizability ofthe particles and the suspending medium, dielectric particles will moveeither toward the regions of high field strength or low field strength.For example, the polarizability of living cells depends on theircomposition, morphology, and phenotype and is highly dependent on thefrequency of the applied electrical field. Thus, cells of differenttypes and in different physiological states generally possess distinctlydifferent dielectric properties, which may provide a basis for cellseparation, e.g., by differential dielecrophoretic forces. Likewise, thepolarizability of droplets also depends upon their size, shape andcomposition. For example, droplets that contain salts can be polarized.According to formulas provided in Fiedler, et al. Analytical Chemistry70, 1909-1915 (1998), individual manipulation of sine, droplets requiresfield differences (inhomogeneities) with dimensions close to thedroplets.

The term “dielectrophoretic force gradient” means a dielectrophoreticforce is exerted on an object in an electric field provided that theobject has a different dielectric constant than the surrounding media.This force can either pull the object into the region of larger field orpush it out of the region of larger field. The force is attractive orrepulsive depending respectively on whether the object or thesurrounding media has the larger dielectric constant.

Manipulation is also dependent on permittivity (a dielectric property)of the droplets and/or particles with the suspending medium. Thus,polymer particles, living cells show negative—dielectrophoresis athigh-field frequencies in water. For example, dielectrophoretic forcesexperienced by a latex sphere in a 0.5 MV/m field (10 V for a 20 micronelectrode gap) in water are predicted to be about 0.2 piconewtons (pN)for a 3.4 micron latex sphere to 15 pN for a 15 micron latex sphere(Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998)). Thesevalues are mostly greater than the hydrodynamic forces experienced bythe sphere in a stream (about 0.3 pN for a 3.4 micron sphere and 1.5 pNfor a 15 micron sphere). Therefore, manipulation of individual cells orparticles can be accomplished in a streaming fluid, such as in a cellsorter device, using dielectrophoresis. Using conventional semiconductortechnologies, electrodes can be microfabricated onto a substrate tocontrol the force fields in a microfabricated sorting device of theinvention. Dielectrophoresis is particularly suitable for moving objectsthat are electrical conductors. The use of AC current is preferred, toprevent permanent alignment of ions. Megahertz frequencies are suitableto provide a net alignment, attractive force, and motion over relativelylong distances. See U.S. Pat. No. 5,454,472.

Radiation pressure can also be used in the invention to deflect and moveobjects, e.g. droplets and particles (molecules, cells, particles, etc.)contained therein, with focused beams of light such as lasers. Flow canalso be obtained and controlled by providing a pressure differential orgradient between one or more channels of a device or in a method of theinvention.

Molecules, cells or particles (or droplets containing molecules, cellsor particles) can be moved by direct mechanical switching, e.g., withon-off valves or by squeezing the channels. Pressure control may also beused, for example, by raising or lowering an output well to change thepressure inside the channels on the chip. See, e.g., the devices andmethods described U.S. Pat. No. 6,540,895. These methods and devices canfurther be used in combination with the methods and devices described inpending U.S. Patent Application Publication No. 20010029983 and20050226742. Different switching and flow control mechanisms can becombined on one chip or in one device and can work independently ortogether as desired.

Inlet Module

The microfluidic device of the present invention includes one or moreinlet modules. An “inlet module” is an area of a microfluidic substratedevice that receives molecules, cells, small molecules or particles foradditional coalescence, detection and/or sorting. The inlet module cancontain one or more inlet channels, wells or reservoirs, openings, andother features which facilitate the entry of molecules, cells, smallmolecules or particles into the substrate. A substrate may contain morethan one inlet module if desired. Different sample inlet channels cancommunicate with the main channel at different inlet modules.Alternately, different sample inlet channels can communication with themain channel at the same inlet module. The inlet module is in fluidcommunication with the main channel. The inlet module generallycomprises a junction between the sample inlet channel and the mainchannel such that a solution of a sample (i.e., a fluid containing asample such as molecules, cells, small molecules (organic or inorganic)or particles) is introduced to the main channel and forms a plurality ofdroplets. The sample solution can be pressurized. The sample inletchannel can intersect the main channel such that the sample solution isintroduced into the main channel at an angle perpendicular to a streamof fluid passing through the main channel. For example, the sample inletchannel and main channel intercept at a T-shaped junction; i.e., suchthat the sample inlet channel is perpendicular (90 degrees) to the mainchannel. However, the sample inlet channel can intercept the mainchannel at any angle, and need not introduce the sample fluid to thechannel at an angle that is perpendicular to that flow. The anglebetween intersecting channels is in the range of from about 60 to about120 degrees. Particular exemplary angles are 45, 60, 90, and 120degrees.

Embodiments of the invention are also provided in which there are two ormore inlet modules introducing droplets of samples into the mainchannel. For example, a first inlet module may introduce droplets of afirst sample into a flow of fluid in the main channel and a second inletmodule may introduce droplets of a second sample into the flow of fluidin main channel, and so forth. The second inlet module is preferablydownstream from the first inlet module (e.g., about 30 μm). The fluidsintroduced into the two or more different inlet modules can comprise thesame fluid or the same type of fluid (e.g., different aqueoussolutions). For example, droplets of an aqueous solution containing anenzyme are introduced into the main channel at the first inlet moduleand droplets of aqueous solution containing a substrate for the enzymeare introduced into the main channel at the second inlet module.Alternatively, the droplets introduced at the different inlet modulesmay be droplets of different fluids which may be compatible orincompatible. For example, the different droplets may be differentaqueous solutions, or droplets introduced at a first inlet module may bedroplets of one fluid (e.g., an aqueous solution) whereas dropletsintroduced at a second inlet module may be another fluid (e.g., alcoholor oil).

Droplet Interdigitation

Particular design embodiments of the microfluidic device describedherein allow for a more reproducible and controllable interdigitation ofdroplets of specific liquids followed by pair-wise coalescence of thesedroplets, described in further detail herein. The droplet pairs cancontain liquids of different compositions and/or volumes, which wouldthen combine to allow for a specific reaction to be investigated. Thepair of droplets can come from any of the following: (i) two continuousaqueois streams and an oil stream; (ii) a continuous aqueous stream, anemulsion stream, and an oil stream, or (iii) two emulsion streams and anoil stream. The term “interdigitation” as used herein means pairing ofdroplets from separate aqueous streams, or from two separate inletnozzles, for eventual coalescence.

The nozzle designs described herein enhance the interdigitation ofdroplets and further improves coalescence of droplets due to the bettercontrol of the interdigitation and smaller distance between pairs ofdroplets. The greater control over interdigitation allows for a perfectcontrol over the frequency of either of the droplets. To obtain theoptimum operation, the spacing between droplets and coupling of thedroplets can be adjusted by adjusting flow of any of the streams,viscosity of the streams, nozzle design (including orifice diameter, thechannel angle, and post-orifice neck of the nozzle

Reservoir/Well

A device of the invention can include a sample solution reservoir orwell or other apparatus for introducing a sample to the device, at theinlet module, which is typically in fluid communication with inletchannel. Reservoirs and wells used for loading one or more samples ontothe microfluidic device of the present invention, include but are notlimited to, syringes, cartridges, vials, eppendorf tubes and cellculture materials (e.g., 96 well plates). A reservoir may facilitateintroduction of molecules or cells into the device and into the sampleinlet channel of each analysis unit.

Fluidic Interconnects

The microfluidic device can include a syringe (or other glass container)that is treated with a vapor or solution of an appropriate PEG-silane toeffect the surface PEG functionalization. The purpose for treating thewalls of glass containers (e.g., syringes) with a PEG functionality isto prevent biological adhesion to the inner walls of the container,which frustrates the proper transfer of biological/chemical materialsinto the microfluidic device of the present invention. The inlet channelis further connected to a means for introducing a sample to said device.The means can be a well or reservoir. The means can be temperaturecontrolled. The inlet module may also contain a connector adapted toreceive a suitable piece of tubing, such as liquid chromatography orHPLC tubing, through which a sample may be supplied. Such an arrangementfacilitates introducing the sample solution under positive pressure inorder to achieve a desired infusion rate at the inlet module.

The interconnections, including tubes, must be extremely clean and makeexcellent bonding with the PDMS surface in order to allow properoperation of the device. The difficulty in making a fluidic! connectionto a microfluidic device is primarily due to the difficulty intransitioning from a macroscopic fluid line into the device whileminimizing dead volume.

In order to minimize contamination and leakage and allow for greaterreproducibility and reliability are improved, tubes and interconnectsfor the PDMS slab can be cured in place. The tubes and interconnects canbe placed in position by applying a UV-cured adhesive to allow forholding the tubes in place on the silicone wafer. Once the tubes areplaced in position, PDMS can be poured over the wafer and cured. Thecured PDMS, along with the tubes in place, can be peeled off of thesilicone wafer easily. This process can be applied to fluidics channelsas well as other connection channels. Once the adhesive is applied ontothe wafer, the process will allow for quick templating of PDMS slabswith exact reproducibility of channel locations and cleanliness. Tubesof any size can be implemented for this process. This process allows forless stress on the interconnection joints and smaller interconnectionfootprints in the device (see, for example, PCT/US2006/02186 filed onJun. 1, 2006; PCT/US2006/021280 filed on Jun. 1, 2006 andPCT/US2006/021380 filed on Jun. 1, 2006, each of which is incorporatedby reference in their entirety for all purposes).

The tubing side of the interconnect can be mounted into a retainingblock that provides precise registration of the tubing, while themicrofluidic device can be positioned accurately in a carrier that theretaining block would align and clamp to. The total dead volumeassociated with these designs would be critically dependent on howaccurately the two mating surfaces could be positioned relative to eachother. The maximum force required to maintain the seal would be limitedby the exact shape and composition of the sealing materials as well asthe rigidity and strength of the device itself. The shapes of the matingsurfaces can be tailored to the minimal leakage potential, sealing forcerequired, and potential for misalignment. By way of non-limitingexample, the single ring indicated in can be replaced with a series ofrings of appropriate cross-sectional shape.

Reservoirs and wells used for loading one or more samples onto themicrofluidic device of the present invention, include but are notlimited to, syringes, cartridges, vials, eppendorf tubes and cellculture materials (e.g., 96 well plates) as described above. One of theissues to be resolved in loading samples into the inlet channel at theinlet module of the substrate is the size difference between the loadingmeans or injection means, e.g., capillary or HPLC tubing and the inletchannel. It is necessary to create an interconnect and loading methodwhich limits leaks and minimizes dead volume and compliance problems.Several devices and methods described in further detail herein addressand solve these art problems.

Self-Aligning Fluidic Interconnects

The present invention includes one or more inlet modules comprisingself-aligning fluidic interconnects proximate to one or more inletchannels to improve the efficiency of sample loading and/or injection.

The present invention proposes the use of small interconnects based oncreating a radial seal instead of a face seal between the microfluidicdevice and interconnect. The inserted interconnect would have a largerdiameter than the mating feature on the device. When inserted, thestretching of the chip would provide the sealing force needed to make aleak-free seal between the external fluid lines and the microfluidicdevice. FIG. 11 details design possibilities for making this seal.

Studies were performed with the leftmost design of FIG. 1 using a casthole in PDMS and 1/32″ PEEK tubing, which showed that the seal was ableto withstand more than 90 PSI of pressure without leakage.

In order to handle instrument and chip manufacturing tolerances, theexternal interconnect must be self aligning and the “capture radius” ofthe molded hole must be large enough to reliably steer the interconnectto the sealing surfaces. FIG. 12 shows that the entrance to the moldedhole is large enough to guarantee capture but tapers down to the sealingsurfaces. The external interconnect could be made directly out of thetubing leading up to the microfluidic substrate, thus eliminatingpotential leak points and unswept volumes. As seen in FIG. 12, theinterconnect is surrounded by the substrate interconnects or “chip dock”for most of its length to make certain it is held within the tolerancestack-up of the system. The external interconnect is made from a hardbut flexible material such as 1/32″ PEEK tubing. The features in themicrofluidic device can be molded directly into it during themanufacturing process, while the inserted seals can be molded/machineddirectly onto the tubing ends or molded as individual pieces andmechanically fastened to the tubing. The retaining ferrule shown in FIG.12 would be attached during manufacturing and provide good absolutereferencing of the tube length. The ferrule could be an off-the-shelfcomponent or a custom manufactured part and be made from, for example, apolymer, an elastomer, or a metal. The tubing end could be tapered onthe end (top most diagram) or squared off (the figure above). Thespecific shape of the end will be controlled by how easily themicrofluidic device will gall during insertion.

Alternatively, it is also possible to mold all the interconnects neededfor each tube into a single monolithic self-aligned part as detailed inFIG. 13. This may help reduce the difficulty in maintaining alignment ofmany external fluidic lines to the chip.

Methods for Molding Fluidic Interconnects Directly on the Substrate

The present invention also provides methods of direct molding of fluidicinterconnects into a microfluidic device. Development of a commercialmicrofluidic platform requires a simple, reliable fluidic interconnectin order to reduce the chance of operator error and leaks. Molding theseinterconnects directly into the microfluidic device requires precisealignment of the molding pins to 30 the patterned shim (the “master”manufactured from Silicon/photoresist or made from some metal) used toform the microfluidic and electrical channels. The extreme tolerancesrequired when molding with low viscosity elastomer such as PDMS requiresnear perfect sealing of the pin face to the master, while stillaccommodating imperfections in the master and assembly of the moldingtool. In an embodiment, the present invention provides a precise andrepeatable method of molding of interconnects while accommodating theimperfections in the molding process by introducing movable pinscaptured in an elastomeric sleeve molded directly into the tool. Inorder to effectively produce at relatively low volume and be able toinexpensively prototype devices, the tool must be able to use mastersgenerated using standard photolithographic processes (e.g. siliconwafers patterned with SU¬18).

FIG. 14 shows a schematic of a molding tool based on this concept. InFIG. 14, the pins (orange) are captured within an elestomeric moldedsleeve. A compression plate made from a rigid backer plate and foamrubber is used to apply gentle even pressure to the pins and generatethe force needed to make the pins uniformly contact the master. Themolded sleeve was found to be necessary to consistently prevent theuncured elastomer from penetrating the region between the pin and thetop plate. Early designs used pins captured in tight clearance holes,and the pins would frequently bind in place (even with lubricant),preventing smooth motion of the pins and improper contact with themaster. This would in turn cause a thin film of the elastomer to formbetween the bottom of the pin and the master (“Flash”). This flashprevents proper operation of the interconnects during chip operation.The addition of the elastomeric sleeves around each pin eliminated thisproblem, and produce consistent, reliable shutoff between the master andthe pins.

Acoustic Actuator

The well or reservoir of the inlet module further include an acousticactuator. To obtain one droplet comprising a single element of aspecific biological/chemical material (e.g., a cell), separation ofbiological/chemical material, and uniformity of the number density ofbiological/chemical materials in a microfluidic channel is desirable.Accordingly, the microfluidic device can include an acoustic actuator.The loaded sample (biological/chemical material) can be well mixed andseparated in a small chamber by acoustic wave before sending out to thenozzle region for encapsulation. The frequency of the acoustic waveshould be fine tuned so as not to cause any damage to the cells. Thebiological effects of acoustic mixing have been well studied (e.g., inthe ink-jet industry) and many published literatures also showed thatpiezoelectric microfluidic device can deliver intact biological payloadssuch as live microorganisms and DNA.

The design of the acoustic resonant can use a Piezoelectric bimorph flatplate located on the side of the carved resonant in the PDMS slab. Theresonant inlet can connect to the cell flow input channel and the outletcan connect to the cell flow pinching channel. The piezoelectric drivingway form can be carefully optimized to select the critical frequenciesthat can separate cells in fluids. There are five parameters to optimizebeyond the frequency parameter and Lab electronics can be used tooptimize the piezoelectric driving waveform. Afterwards, a low costcircuit can be designed to generate only the optimized waveform in apreferred microfluidic device.

Coalescence Module

The microfluidic device of the present invention also includes one ormore coalescence modules. A “coalescence module” is within or coincidentwith at least a portion of the main channel at or downstream of theinlet module where molecules, cells, small molecules or particlescomprised within droplets are brought within proximity of other dropletscomprising molecules, cells, small molecules or particles and where thedroplets in proximity fuse, coalesce or combine their contents. Thecoalescence module can also include an apparatus, for generating anelectric force.

The electric force exerted on the fluidic droplet may be large enough tocause the droplet to move within the liquid. In some cases, the electricforce exerted on the fluidic droplet may be used to direct a desiredmotion of the droplet within the liquid, for example, to or within achannel or a microfluidic channel (e.g., as further described herein),etc.

The electric field can be generated from an electric field generator,i.e., a device or system able to create an electric field that can beapplied to the fluid. The electric field generator may produce an ACfield (i.e., one that varies periodically with respect to time, forexample, sinusoidally, sawtooth, square, etc.), a DC field (i.e., onethat is constant with respect to time), a pulsed field, etc. Theelectric field generator may be constructed and arranged to create anelectric field within a fluid contained with a channel or a microfluidicchannel. The electric field generator may be integral to or separatefrom the fluidic system containing the channel or microfluidic channel,according to some embodiments. As used herein, “integral” means thatportions of the components integral to each other are joined in such away that the components cannot be in manually separated from each otherwithout cutting or breaking at least one of the components.

Techniques for producing a suitable electric field (which may be AC, DC,etc.) are known to those of ordinary skill in the art. For example, inone embodiment, an electric field is produced by applying voltage acrossa pair of electrodes, which may be positioned on or embedded within thefluidic system (for example, within a substrate defining the channel ormicrofluidic channel), and/or positioned proximate the fluid such thatat least a portion of the electric field interacts with the fluid. Theelectrodes can be fashioned from any suitable electrode material ormaterials known to those of ordinary skill in the art, including, butnot limited to, silver, gold, copper, carbon, platinum, copper,tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as wellas combinations thereof.

Electrodes

The device can include channels for use in fluid control and otherchannels filled with a metal alloy for casting integrated metal alloycomponents (i.e., electrodes). Alternatively, the electrodes can bemanufactured using other technologies (e.g., lithographically patternedelectrodes made from indium tin oxide or a metal such as platinum). Themicrofluidic device can include metal alloy components useful forperforming electrical functions on fluids, including but not limited to,coalescing droplets, charging droplets, sorting droplets, detectingdroplets and shaking droplets to mix the contents of coalesced droplets.The device can contain more than one of the above mentioned componentsfor more than one of the above mentioned functions.

The electrodes comprising metal alloy components may either terminate atfluid channels or be isolated from fluid channels. The electrodes can beconstructed by filling the appropriate channels with metal alloy. Oneway this can be accomplished is to use positive pressure injection ofthe metal alloy in a melted state, such as with a syringe, into thechannels, and then cool the metal alloy to a solid form. Another exampleis to use negative pressure to draw the metal alloy in a melted stateinto the channels, and then cool the metal alloy to a solid form. Thiscan be accomplished for example by use of capillary forces. Anothermethod of construction can use any of the above mentioned embodiments,and then flush out the metal alloy in a melted state with another liquidto define the geometry of the metal alloy components. Another example isto use any of the above mentioned embodiments, and then use a localizedcold probe to define a solid termination point for the metal alloy, andthen cool the remaining metal alloy to a solid form. A further exampleis to use another material, such as microscopic solder spheres or UVcurable conductive ink, to form a barrier between fluid and metal alloychannels, to define the geometry of the metal alloy components.

The device can include a combination of both integrated metal alloycomponents and a patterned electrically conductive layer. The patternedelectrically conductive layer can have features patterned such thattheir boundaries are within a leak-proof seal. The device can have apatterned electrically conductive feature as one of two chargingelectrodes and one integrated metal alloy component as the other of twocharging electrodes.

The device can include a plurality of electrodes that are insulated fromthe fluid present in the device, and the method of operation includingappropriate application of dielectrical signals and appropriate fluids.In known devices, the electrodes are typically in contact with thefluids in order to allow discharge of species that would otherwisescreen the applied dielectric field. Whereas, in devices where theelectrodes have been insulated from the fluid, this screening effecttypically arises so quickly that the device is not useful for anysignificantly extended period of time. The drawbacks of electrodes incontact with the fluids vs. insulated electrodes are (a) degradedreliability against leaking (since the interface between the electrodesand the other components of the device may be more difficult to effect aleak-proof seal), and (b) degraded reliability against electrodecorrosion (whose failure mode effects include failure of application ofdielectric fields, and fluid channel contamination).

The device of the present invention comprising a plurality of electrodesthat are insulated from the fluid present in the device counteracts thisscreening effect by extending the screening rise time and including apolarity switch for all of the different dielectric fields applied inthe device. The screening rise time is extended by using fluids withdielectrical properties. A polarity switch for all of the differentdielectric fields applied in the device is achieved by using analgorithm for dielectrical control, which switches the polarity of thedielectrical fields at a frequency sufficiently high to maintain properdielectrical function of the device. This dielectrical control algorithmmay also switch the polarity for the dielectric fields in a cascading,time controlled manner starting at the fluid origin point andprogressing downstream, so that given fluid components experience onepolarity at every point along their course. The device of the presentinvention can be used with metal alloy electrodes or using a combinationof metal alloy electrodes and patterned conductive film electrodes.

The invention can provide a microfluidic device using injectedelectrodes. The interface between the microscopic electrode (typically25 μm thick) and the macroscopic interconnect can easily fail if thejoint between the two is flexed. The flexing of the joint can beeliminated by securing a firm material that serves to fasten, support,and re-enforce the joint (i.e., a grommet) into the interface. In orderto prevent flexing, the mating surface of the device can be manufacturedfrom a hard material such as glass or plastic. The electrical connectionwith the external system can be made by securing the device such that itconnects to a spring loaded contact, which is either offset from thegrommet (thereby minimizing the force applied to the solder region), orcentered on the grommet (as long as the contact does not touch thesolder).

The metal alloy components are also useful for performing opticalfunctions on fluids, including but not limited to, optical detection ofdroplets in a geometry which may include a mirror.

To prevent leakage of fluid out of electrodes placed within microfluidicchannels, the microfluidic device can include a layer patterned withchannels for fluid control, and another layer with patternedelectrically conductive features, where the features are patterned suchthat their boundaries are within a leak-proof seal. The leak-proof sealcan be achieved at the interface between the unpatterned areas of thefluid control layer and the unpatterned areas of the electricallyconductive layer. The leak-proof seal can also be achieved by a thirdinterfacial layer between the fluid control layer and the unpatternedareas of the electrically conductive layer. The third interfacial layercan or can not be perforated at specific locations to allow contactbetween the fluid and the electrically conductive layer. Electricalaccess ports can also be patterned in the fluid control layer.

The electrodes and patterned electrically conductive layers as describedcan be associated with any module of the device (inlet module,coalescence module, mixing module, delay module, detection module andsorting module) to generate dielectric or electric forces to manipulateand control the droplets and their contents.

Effective control of uncharged droplets within microfluidic devices canrequire the generation of extremely strong dielectric field gradients.The fringe fields from the edges of a parallel plate capacitator canprovide an excellent topology to form these gradients. The microfluidicdevice according to the present invention can include placing a fluidicchannel between two parallel electrodes, which can result in a steepelectric field gradient at the entrance to the electrodes due to edgeeffects at the ends of the electrode pair. Placing these pairs ofelectrodes at a symmetric channel split can allow precise bi-directionalcontrol of droplet within a device. Using the same principle, only withasymmetric splits, can allow single ended control of the dropletdirection in the same manner. Alternatively, a variation on thisgeometry will allow precise control of the droplet phase by shifting.

In some cases, transparent or substantially transparent electrodes canbe used. The electric field generator can be constructed and arranged(e.g., positioned) to create an electric field applicable to the fluidof at least about 0.01 V/micrometer, and, in some cases, at least about0.03 V/micrometer, at least about 0.05 V/micrometer, at least about 0.08V/micrometer, at least about 0.1 V/micrometer, at least about 0.3V/micrometer, at least about 0.5 V/micrometer, at least about 0.7V/micrometer, at least about 1 V/micrometer, at least about 1.2V/micrometer, at least about 1.4 V/micrometer, at least about 1.6V/micrometer, or at least about 2 V/micrometer. In some embodiments,even higher electric field intensities may be used, for example, atleast about 2 V/micrometer, at least about 3 V/micrometer, at leastabout 5 V/micrometer, at least about 7 V/micrometer, or at least about10 V/micrometer or more.

As described, an electric field may be applied to fluidic droplets tocause the droplets to experience an electric force. The electric forceexerted on the fluidic droplets may be, in some cases, at least about10⁻¹⁶N/micrometer³. In certain cases, the electric force exerted on thefluidic droplets may be greater, e.g., at least about 10⁻¹⁵N/micrometer³, at least about 10⁻¹⁴ N/micrometer³, at least about10⁻¹³N/micrometer³, at least about 10⁻¹² N/micrometer³, at least about10⁻¹¹ N/micrometer³, at least about 10⁻¹⁰ N/micrometer3, at least about10⁻⁹N/micrometer³, at least about 10⁻⁸ N/micrometer³, or at least about10⁻⁷ N/micrometer³ or more. The electric force exerted on the fluidicdroplets, relative to the surface area of the fluid, may be at leastabout 10⁻¹⁵ N/micrometer², and in some cases, at least about 10⁻¹⁴N/micrometer², at least about 10⁻¹³ N/micrometer², at least about 10⁻¹²N/micrometer², at least about 10⁻¹¹ N/micrometer², at least about 10⁻¹⁰N/micrometer², at least about 10⁻⁹ N/micrometer², at least about 10⁻⁸N/micrometer², at least about 10⁻⁷ N/micrometer², or at least about 10⁻⁶N/micrometer² or more. In yet other embodiments, the electric forceexerted on the fluidic droplets may be at least about 10⁻⁹ N, at leastabout 10⁻⁸ N, at least about 10⁻⁷ N, at least about 10⁻⁶N, at leastabout 10⁻⁵ N, or at least about 10⁻⁴ N or more in some cases.

Channel Expansion Geometries

In preferred embodiments described herein, droplet coalescence ispresently carried out by having two droplet forming nozzles emittingdroplets into the same main channel. The size of the nozzles allow forone nozzle to form a large drop that fills the exhaust line while theother nozzle forms a drop that is smaller than the first. The smallerdroplet is formed at a rate that is less than the larger droplet rate,which insures that at most one small droplet is between big droplets.Normally, the small droplet will catch up to the larger one over arelatively short distance, but sometimes the recirculation zone behindthe large drop causes the small drop to separate from the large dropcyclically. In addition, the small drop occasionally does not catch upwith the large one over the distance between the nozzles and thecoalescing electrodes. Thus, in some situations is a need for a morerobust coalescence scheme.

Geometric alterations in the coalescence module can create a morerobust, reliable coalescence or fusing of droplets over a wider range ofsizes and flows. The solution to improve the performance is to place anexpansion in the main channel between the electrodes. FIG. 15 is aschematic diagram of the improved coalescence module. Optionally, asmall constriction (neckdown) just before this expansion can be used tobetter align the droplets on their way into the coalescence point (alsoshown in the FIG. 15). This optional neckdown can help center the smalldroplet in the channel stream lines, reducing the chance that it willflow around the larger droplet prior to coalescing in the expansion. Theelectrode pair may be placed on either one side of the channel or onboth sides.

The expansion in the coalescing region allows for a dramatic catching upof the small drop to the large drop, as shown through micrographs takenon an operating device. The volume of the expansion is big enough toslow the large droplet down so that the small drop always catches up tothe large drop, but doesn't allow the next large drop to catch up andmake contact with the pair to be coalesced. The electrodes allow forcoalescence to take place when the drops are in contact with each otherand passing through the field gradient.

Detection Module

The microfluidic device of the present invention can also include one ormore detection modules. A “detection module” is a location within thedevice, typically within the main channel where molecules, cells, smallmolecules or particles are to be detected, identified, measured orinterrogated on the basis of at least one predetermined characteristic.The molecules, cells, small molecules or particles can be examined oneat a time, and the characteristic is detected or measured optically, forexample, by testing for the presence or amount of a reporter. Forexample, the detection module is in communication with one or moredetection apparatuses. The detection apparatuses can be optical orelectrical detectors or combinations thereof. Examples of suitabledetection apparatuses include optical waveguides, microscopes, diodes,light stimulating devices, (e.g., lasers), photo multiplier tubes, andprocessors (e.g., computers and software), and combinations thereof,which cooperate to detect a signal representative of a characteristic,marker, or reporter, and to determine and direct the measurement or thesorting action at the sorting module. However, other detectiontechniques can also be employed

The term “determining,” as used herein, generally refers to the analysisor measurement of a species, for example, quantitatively orqualitatively, and/or the detection of the presence or absence ofspecies. “Determining” may also refer to the analysis or measurement ofan interaction between more species, for example, quantitatively orqualitatively, or by detecting the presence or absence of theinteraction. Examples of suitable techniques include, but are notlimited to, spectroscopy such as infrared, absorption, fluorescence,UV/visible, FTIR (“Fourier Transform Infrared Spectroscopy”), or Raman;gravimetric techniques; ellipsometry; piezoelectric measurements;immunoassays; electrochemical measurements; optical measurements such asoptical density measurements; circular dichroism; light scatteringmeasurements such as quasielectric light scattering; polarimetry;refractometry; or turbidity measurements as described further herein.

A detection module is within, communicating or coincident with a portionof the main channel at or downstream of the inlet module and, in sortingembodiments, at, proximate to, or upstream of, the sorting module orbranch point. The sorting module may be located immediately downstreamof the detection module or it may be separated by a suitable distanceconsistent with the size of the molecules, the channel dimensions andthe detection system. Precise boundaries for the detection module arenot required, but are preferred.

Detection modules used for detecting molecules and cells have across-sectional area large enough to allow a desired molecule, cells,bead, or particles to pass through without being substantially sloweddown relative to the flow carrying it. The dimensions of the detectionmodule are influenced by the nature of the sample under study and, inparticular, by the size of the droplets, beads, particles, molecules orcells (including virions) under study. For example, mammalian cells canhave a diameter of about 1 to 50 microns, more typically 10 to 30microns, although some mammalian cells (e.g., fat cells) can be largerthan 120 microns. Plant cells are generally 10 to 100 microns. However,other molecules or particles can be smaller with a diameter from about20 nm to about 500 nm.

Waveguides

The present invention provides self-aligning optical waveguides andoptical elements (lenses, mirrors, interconnects, etc.) for detectionand control of droplets. Such waveguides can be provide well definedoptical access to the fluidic channels to permit optical scattering,absorption, fluorescence, or any other optical measurement technique.

In order to create the waveguides, a separate series of channels anduseful shapes (lenses, mirrors, etc) can be created eithersimultaneously within the other channels in the substrate (i.e. in thesame processing step) or in successive steps. The reusable mastercreated in this way can then used to form the waveguide components andfluid channels without the need for special fixturing or carefulshipment in subsequent steps. The extra channels or shapes can thenfilled with a high index of refraction liquid (for waveguides) orreflective material (for mirrors) through injection into the channel orvoid. The liquid can either remain as a fluid or be allowed to solidify.UV cure epoxies used by the telecommunications industry are excellentchoices for the waveguide materials. Possible waveguide geometry caninclude a focusing lens and a back-reflecting mirror.

Sensors

One or more detections sensors and/or processors may be positioned to bein sensing communication with the fluidic droplet. “Sensingcommunication,” as used herein, means that the sensor may be positionedanywhere such that the fluidic droplet within the fluidic system (e.g.,within a channel), and/or a portion of the fluidic system containing thefluidic droplet may be sensed and/or determined in some fashion. Forexample, the sensor may be in sensing communication with the fluidicdroplet and/or the portion of the fluidic system containing the fluidicdroplet fluidly, optically or visually, thermally, pneumatically,electronically, or the like. The sensor can be positioned proximate thefluidic system, for example, embedded within or integrally connected toa wall of a channel, or positioned separately from the fluidic systembut with physical, electrical, and/or optical communication with thefluidic system so as to be able to sense and/or determine the fluidicdroplet and/or a portion of the fluidic system containing the fluidicdroplet (e.g., a channel or a microchannel, a liquid containing thefluidic droplet, etc.). For example, a sensor may be free of anyphysical connection with a channel containing a droplet, but may bepositioned so as to detect electromagnetic radiation arising from thedroplet or the fluidic system, such as infrared, ultraviolet, or visiblelight. The electromagnetic radiation may be produced by the droplet,and/or may arise from other portions of the fluidic system (orexternally of the fluidic system) and interact with the fluidic dropletand/or the portion of the fluidic system containing the fluidic dropletin such as a manner as to indicate one or more characteristics of thefluidic droplet, for example, through absorption, reflection,diffraction, refraction, fluorescence, phosphorescence, changes inpolarity, phase changes, changes with respect to time, etc. As anexample, a laser may be directed towards the fluidic droplet and/or theliquid surrounding the fluidic droplet, and the fluorescence of thefluidic droplet and/or the surrounding liquid may be determined.“Sensing communication,” as used herein may also be direct or indirect.As an example, light from the fluidic droplet may be directed to asensor, or directed first through a fiber optic system, a waveguide,etc., before being directed to a sensor.

Non-limiting examples of detection sensors useful in the inventioninclude optical or electromagnetically-based systems. For example, thesensor may be a fluorescence sensor (e.g., stimulated by a laser), amicroscopy system (which may include a camera or other recordingdevice), or the like. As another example, the sensor may be anelectronic sensor, e.g., a sensor able to determine an electric field orother electrical characteristic. For example, the sensor may detectcapacitance, inductance, etc., of a fluidic droplet and/or the portionof the fluidic system containing the fluidic droplet. In some cases, thesensor may be connected to a processor, which in turn, cause anoperation to be performed on the fluidic droplet, for example, bysorting the droplet.

Characteristics

Characteristics determinable with respect to the droplet and usable inthe invention can be identified by those of ordinary skill in the art.Non-limiting examples of such characteristics include fluorescence,spectroscopy (e.g., optical, infrared, ultraviolet, etc.),radioactivity, mass, volume, density, temperature, viscosity, pH,concentration of a substance, such as a biological substance (e.g., aprotein, a nucleic acid, etc.), or the like.

A corresponding signal is then produced, for example indicating that“yes” the characteristic is present, or “no” it is not. The signal maycorrespond to a characteristic qualitatively or quantitatively. That is,the amount of the signal can be measured and can correspond to thedegree to which a characteristic is present. For example, the strengthof the signal may indicate the size of a molecule, or the potency oramount of an enzyme expressed by a cell, or a positive or negativereaction such as binding or hybridization of one molecule to another, ora chemical reaction of a substrate catalyzed by an enzyme. In responseto the signal, data can be collected and/or a control system in thesorting module, if present, can be activated to divert a droplet intoone branch channel or another for delivery to the collection module orwaste module. Thus, in sorting embodiments, molecules or cells within adroplet at a sorting module can be sorted into an appropriate branchchannel according to a signal produced by the corresponding examinationat a detection module. The means of changing the flow path can beaccomplished through mechanical, electrical, optical, or some othertechnique as described herein.

A preferred detector is an optical detector, such as a microscope, whichmay be coupled with a computer and/or other image processing orenhancement devices to process images or information produced by themicroscope using known techniques. For example, molecules can beanalyzed and/or sorted by size or molecular weight. Enzymes can beanalyzed and/or sorted by the extent to which they catalyze chemicalreaction of a substrate (conversely, substrate can be analyzed and/orsorted by the level of chemical reactivity catalyzed by an enzyme).Cells can be sorted according to whether they contain or produce aparticular protein, by using an optical detector to examine each cellfor an optic indication of the presence or amount of that protein. Theprotein may itself be detectable, for example by a characteristicfluorescence, or it may be labeled or associated with a reporter thatproduces a detectable signal when the desired protein is present, or ispresent in at least a threshold amount. There is no limit to the kind ornumber of characteristics that can be identified or measured using thetechniques of the invention, which include without limitation surfacecharacteristics of the cell and intracellular characteristics, providedonly that the characteristic or characteristics of interest for sortingcan be sufficiently identified and detected or measured to distinguishcells having the desired characteristic(s) from those which do not. Forexample, any label or reporter as described herein can be used as thebasis for analyzing and/or sorting molecules or cells, i.e. detectingmolecules or cells to be collected.

Fluorescence Polarization

As described herein, the biological/chemical entity to be analyzed mayitself be detectable, for example by a characteristic fluorescence, orit may be labeled or associated with a reporter that produces adetectable signal when the desired protein is present, or is present inat least a threshold amount.

Luminescent colloidal semiconductor nanocrystals called quantum dots orq-dots (QD) are inorganic fluorophores that have the potential tocircumvent some of the functional limitations encountered by organicdyes. In particular, CdSe—ZnS core-shell QDs exhibit size-dependenttunable photoluminescence (PL) with narrow emission bandwidths (FWHM ˜30to 45 nm) that span the visible spectrum and broad absorption bands.These allow simultaneous excitation of several particle sizes (colors)at a common wavelength. This, in turn, allows simultaneous resolution ofseveral colors using standard instrumentation. CdSe—ZnS QDs also havehigh quantum yields, are resistant to photodegradation, and can bedetected optically at concentrations comparable to organic dyes.

Quantum dots are nano-scale semiconductors typically consisting ofmaterials such as crystalline cadmium selenide. The term ‘q-dot’emphasizes the quantum confinement effect of these materials, andtypically refers to fluorescent nanocrystals in the quantum confinedsize range. Quantum confinement refers to the light emission from bulk(macroscopic) semiconductors such as LEDs which results from excitingthe semiconductor either electrically or by shining light on it,creating electron-hole pairs which, when they recombine, emit light. Theenergy, and therefore the wavelength, of the emitted light is governedby the composition of the semiconductor material. If, however, thephysical size of the semiconductor is considerably reduced to be muchsmaller than the natural radius of the electron-hole pair (Bohr radius),additional energy is required to “confine” this excitation within thenanoscopic semiconductor structure leading to a shift in the emission toshorter wavelengths. Three different q-dots in several concentrationseach can be placed in a microdroplet, and can then be used with amicrofluidic device to decode what is in the drop. The Q-dot readoutextension to the fluorescence station can be incorporated into thedesign of the microfluidic device. A series of dichroic beamsplitters,emission filters, and detectors can be stacked onto the system, allowingmeasurement of the required five emission channels (two fluorescencepolarization signals and three q-dot bands).

Fluorescence Polarization (FP) detection technology enables homogeneousassays suitable for high throughput screening assays in the DrugDiscovery field. The most common label in the assays is fluorescein. InFP-assay the fluorophore is excited with polarized light. Onlyfluorophores parallel to the light absorb and are excited. The excitedstate has a lifetime before the light emission occurs. this time thelabeled fluorophore molecule rotates and the polarization of the lightemitted differs from the excitation plane. To evaluate the polarizationtwo measurements are needed: the first using a polarized emission filterparallel to the excitation filter (S-plane) and the second with apolarized emission filter perpendicular to the excitation filter(P-plane). The Fluorescence Polarization response is given as mP(milli-Polarization level) and is obtained from the equation:

Polarization (mP)=1000*(S−G*P)/(S+G*P)

Where S and P are background subtracted fluorescence count rates and G(grating) is an instrument and assay dependent factor.

The rotational speed of a molecule is dependent on the size of themolecule, temperature and viscosity of the solution. Fluorescein has afluorescence lifetime suitable for the rotation speeds of molecules inbio-affinity assays like receptor-ligand binding assays or immunoassaysof haptens. The basic principle is that the labeled compound is smalland rotates rapidly (low polarization). When the labeled compound bindsto the larger molecule, its rotation slows down considerably(polarization changes from low to high polarization). Thus, FP providesa direct readout of the extent of tracer binding to protein, nucleicacids, and other biopolymers.

Fluorescence polarization technology has been used in basic research andcommercial diagnostic assays for many decades, but has begun to bewidely used in drug discovery only in the past six years. Originally, FPassays for drug discovery were developed for single-tube analyticalinstruments, but the technology was rapidly converted to high-throughputscreening assays when commercial plate readers with equivalentsensitivity became available. These assays include such well-knownpharmaceutical targets such as kinases, phosphatases, proteases,G-protein coupled receptors, and nuclear receptors. Other homogeneoustechnologies based on fluorescence intensity have been developed. Theseinclude energy transfer, quenching, and enhancement assays. FP offersseveral advantages over these. The assays are usually easier toconstruct, since the tracers do not have to respond to binding byintensity changes. In addition, only one tracer is required and crudereceptor preparations may be utilized. Furthermore, since FP isindependent of intensity, it is relatively immune to colored solutionsand cloudy suspensions. FP offers several advantages in the area ofinstrumentation. Because FP is a fundamental property of the molecule,and the reagents are stable, little or no standardization is required.FP is relatively insensitive to drift in detector gain settings andlaser power.

The dyes chosen for FP are commonly used in most cell- and enzyme-basedassays and are designated not to overlap significantly with the q-dots.The dyes are evaluated both independently and together with the q-dots(at first off-instrument) to assess the cross-talk. Preferably, theliquid q-dot labels are read outside a spectral wavelength bandcurrently used in FACS analysis and sorting (i.e., the dyes flourescein,Cy3, Cy5, etc). This permits the use of currently-available assays(dependent on these dyes). Using specific q-dots, crosstalk isminimized.

Accordingly, the present invention provides methods to label dropletsand/or nanoreactors formed on a microfluidic device by using only asingle dye code to avoid cross-talk with other dyes during FP.Additionally, the present invention provides methods to create FP dyecodes to label compounds contained within liquids (including dropletsand/or nanoreactors) where the compound is designed to be differentiatedby FP on a microfluidic device. In this manner, dye codes having thesame color, absorption, and emission could be used to label compoundswithin liquids.

In one aspect, the present invention is directed to the use offluorescence polarization to label liquids. Droplets can be labeledusing several means. These labeling means include, but are not limitedto, the use of different dyes, quantum dots, capacitance, opacity, lightscattering, fluorecence intensity (FI), fluorescence lifetime (FL),fluorescence polarization (FP), circular dichroism (CD), fluorescenececorrelation and combinations of all of these previous labeling means.The following disclosure describes the use of FP and FI as a means tolabel droplets on a microfluidic device. In addition, the use of FL as ameans to adjust the overall FP of a solution, and by varying theconcentration of the total FI, to create a 2-dimensional encoding schemeis demonstrated.

In general, molecules that take up more volume will tumble slower than asmaller molecule coupled to the same fluorophore (see FIG. 16). FP isindependent of the concentration of the dye; liquids can have vastlydifferent concentrations of FITC in them yet still have identical FPmeasurements.

In a preferred embodiment, a FP dye is an organic dye that does notinterfere with the assay dye is used. Furthermore, since the totalintensity of the FP dye can be quantified, a second dimension in whichto label the droplet is provided. Thus, one can exploit the differencesin FP to create an encoding scheme of dye within a liquid solution,including droplets. An example is shown in FIG. 17 whereby the dropletsare labeled with 3 differently-sized FITC molecules (i.e., threedifferent droplets contain FITC molecules and FITC coupled to eitherbiotin or streptavidin, respectively). Therefore, in a single dimension,FP can be used to create an encoding scheme. However, the presentinvention can also use Fluorescence Intensity (FI) of the overallsolution to create even more labels in a second dimension. An example oflabeling droplets in 2 dimensions is shown in FIG. 18.

Interestingly, the differences of the fluorescence lifetime (FL) of twodyes with spectral overlap in the detected emission wavelength to changethe overall FP of the combined solution can also be exploited (see FIGS.18 and 19).

Although FIG. 17 discusses the use of multiple compounds to which a dyemolecule is attached to span a range of FP, it is also possible to spanthe range using a high and low molecular weight compound set. Asexemplified by FIG. 19, a dye can be attached to a large compound (forexample streptavidin) and kept at a fixed concentration, to which asmaller compound (for example, a free dye molecule) would be titratedinto the same solution. The FP of the solution can be adjusted to be indiscernable increments from the value of the large molecule to somewhereslightly greater than the FP of the smaller molecule. The [total] dyeintensity can be varied by varying the concentration of the mixture ofthe two dye-attached compounds. By varying total dye concentration andthe FP, two dimensions can be used to generate the FP dye codes(FPcodes). Accordingly, many FPcodes can be generated using only twocompounds.

This could also include use of large fluorescent proteins such as GFPand the phycobiliproteins combined with a smaller molecule.

Examples of dyes commonly used in biological dyes are listed in thetable below.

Excitation Emission Examples of Wavelength Wavelength Compatible Dyes450 500 Cyan 500 483 533 SYBR Green, FAM 523 568 HEX, VIC 558 610 RED610 615 640 RED 640 650 670 CY5

In another aspect, the present invention is directed labeling solidsusing properties other than dye emission and dye concentration. In oneembodiment the solid can include, for example, a bead or location on asolid support or chip. As demonstrated above for liquids, FI and FL canbe two of many dimensions of characteristics used as labels. By way ofnon-limiting example, it is possible to use two dyes with different FLto change the overall FP for a solid such as a bead or other mobilesolid support.

In another embodiment, a linker can be used to couple the dye to thebead. The linker can be varied so as to allow the dye to have differingdegrees of freedom in which to rotate (i.e., tumble). Varying the linkerin this manner can change the FP of the attached dye, which in uniquecombinations can be used as a label. In some embodiments, the beads canbe swollen in organic solvent and the dyes held in place by hydrophobicforces. In this case, the FP, FI, FL methods described above for liquidlabeling can also be used as a means for labeling the beads. A quenchingmolecule can also be used to change the characteristics of a dye. Suchquenching can be continuous or brought about through the interaction ofa molecule, such as a peptide or nucleic acid linker, with differingmeans of bringing molecules together depending on the strength oflinker-internal interaction (e.g., a nucleotide stem loop structure ofvarying lengths).

The reactions analyzed on the virtual, random and non-random arrays(discussed briefly below) can be also increased beyond the two (cy3 andcy5 intensities) commonly used for multiplexing. For example, differentFP, FI, etc can be used as a read-out.

Random array decoding: Beads of the prior art use one or morepre-attached oligonucleotide-coupled beads that are held in place in afiber-optic faceplate (for example, those used by Illumina). The oligoson the beads are decoded using sequential hybridization of a labeledcomplementary oligo. The assay of the prior art uses a separateoligonucleotide complementary zipcode (‘Illumacode’) attached to eachtype of bead.

The invention described herein is superior to the methods of the priorart in that the FP, FI, FL-labeled bead or mobile solid support can beplaced into a random array (e.g., a chip as manufactured by Illumina)and the FP, FI, FL used to decode the bead. The FP, FI, FL of the beadcan be decoded before using the chip and the different beads ‘mapped’ asto their specific locations. Alternatively, the bead can be decodedduring attachment of the assay read-out. Significantly, the methodsdescribed by the present invention can be used to pre-determine thelocation of each bead-type either before, or during analysis.

Virtual array decoding: Methods of the prior art use 2 lasers and 3detectors to differentiate a set of 100 bead-types. The beads-types aredifferentiated by the FI of two different dyes present in 1 of 10concentrations (per dye) contained within the bead, and the assaydetector is used to measure fluorescein concentration on the bead. Thedyes, which are added to organic-solvent swollen beads, are not directlyattached to the beads, but remain held within the bead by hydrophobicforces.

Using the methods of the present invention as described herein, a seconddetector to the machines of the prior art used to measure FP can beadded, thereby adding a third dimension and extending the encodingscheme beyond the 100 available in the prior art.

Non-random array decoding: In chips of the prior art (such as those usedby Affymetrix) oligonucleotides are synthesized directly on the chip.Decoding is simply a matter of knowing the location of the assay on thechip.

The methods as described herein can be advantageously used inconjunction with such chips to increase the number of things that can besimultaneously analyzed (i.e., multiplexed) on the chip. By way ofnon-limiting example, Cy3, Cy5, FL and FP can be used as analysismarkers for hybridization reactions.

The present invention also provides methods for labeling micro ornano-sized droplets using Radio Frequency Identification (RFID). RFIDtags can improve the identification of the contents within the droplets.Preferably, the droplets are utilized within a microfluidic device.

RFID is an automatic identification method, relying on storing andremotely retrieving data using devices called RFID tags or transponders.An RFID tag is an object that can be attached to or incorporated into aproduct, animal, or person for the purpose of identification using radiowaves. Chip-based RFID tags contain silicon chips and antennae. Passivetags require no internal power source, whereas active tags require apower source. Hitachi has “powder” 0.05 mm×0.05 mm RFID chips. The newchips are 64 times smaller than the previous record holder, the 0.4mm×0.4 mm mu-chips, and nine times smaller than Hitachi's last yearprototype, and have room for a 128-bit ROM that can store a unique38-digit ID number.

In one embodiment, a solution containing RFID tags are emulsified intodroplets and are used as a label for the identification of the materialwithin the droplet solution. Applications include, but are not limitedto; genetics, genomics, proteomics, chemical synthesis, biofuels, andothers.

Lasers

To detect a reporter or determine whether a molecule, cell or particlehas a desired characteristic, the detection module may include anapparatus for stimulating a reporter for that characteristic to emitmeasurable light energy, e.g., a light source such as a laser, laserdiode, light emitting diode (LED), high-intensity lamp, (e.g., mercurylamp), and the like. Where a lamp is used, the channels are preferablyshielded from light in all regions except the detection module. Where alaser is used, the laser can be set to scan across a set of detectionmodules from different analysis units. In addition, laser diodes orLED's may be microfabricated into the same chip that contains theanalysis units. Alternatively, laser diodes or LED's may be incorporatedinto a second chip (i.e., a laser diode chip) that is placed adjacent tothe analysis or microchip such that the laser light from the diodesshines on the detection module(s).

An integrated semiconductor laser and/or an integrated photodiodedetector can be included on the substrate in the vicinity of thedetection module. This design provides the advantages of compactness anda shorter optical path for exciting and/or emitted radiation, thusminimizing distortion and losses.

Fluorescence produced by a reporter is excited using a laser beamfocused on molecules (e.g., DNA, protein, enzyme or substrate) or cellspassing through a detection region. Fluorescent reporters can include,but are not limited to, rhodamine, fluorescein, Texas red, Cy 3, Cy 5,phycobiliprotein (e.g., phycoerythrin), green fluorescent protein (GFP),YOYO-1 and PicoGreen. In molecular fingerprinting applications, thereporter labels can be fluorescently labeled single nucleotides, such asfluorescein-dNTP, rhodamine-dNTP, Cy3-dNTP, etc.; where dNTP representsdATP, dTTP, dUTP or dCTP. The reporter can also be chemically-modifiedsingle nucleotides, such as biotin-dNTP. The reporter can befluorescently or chemically labeled amino acids or antibodies (whichbind to a particular antigen, or fragment thereof, when expressed ordisplayed by a cell or virus).

The device can analyze and/or sort cells based on the level ofexpression of selected cell markers, such as cell surface markers, whichhave a detectable reporter bound thereto, in a manner similar to thatcurrently employed using fluorescence-activated cell sorting (FACS)machines. Proteins or other characteristics within a cell, and which donot necessarily appear on the cell surface, can also be identified andused as a basis for sorting. The device can also determine the size ormolecular weight of molecules such as polynucleotides or polypeptides(including enzymes and other proteins) or fragments thereof passingthrough the detection module. Alternatively, the device can determinethe presence or degree of some other characteristic indicated by areporter. If desired, the cells, particles or molecules can be sortedbased on this analysis. The sorted cells, particles or molecules can becollected from the outlet channels in collection modules (or discardedin wasted modules) and used as needed. The collected cells, particles ormolecules can be removed from the device or reintroduced to the devicefor additional coalescence, analysis and sorting.

Processors

As used herein, a “processor” or a “microprocessor” is any component ordevice able to receive a signal from one or more sensors, store thesignal, and/or direct one or more responses (e.g., as described above),for example, by using a mathematical formula or an electronic orcomputational circuit. The signal may be any suitable signal indicativeof the environmental factor determined by the sensor, for example apneumatic signal, an electronic signal, an optical signal, a mechanicalsignal, etc.

The device of the present invention can comprise features, such asintegrated metal alloy components and/or features patterned in anelectrically conductive layer, for detecting droplets by broadcasting asignal around a droplet and picking up an electrical signal in proximityto the droplet.

Parallel Analysis

The droplet content detection can also be achieved by simultaneousdetection of contents of multiple droplets in parallel usingspectroscopic fluorescence imaging with sensitivity as high assingle-molecule limit. One can spatially distribute droplets containingfluorescent entities such as fluorophore biological markers and/orquantum dots in a two-dimensional sheet in a microscopic field-of-view.The field-of-view of those droplets can then be illuminated by afluorescence excitation source and the resulting fluorescence can bespectroscopically imaged. Therefore, for a given fluorescence detectionsensitivity, the throughput of fluorescence detection compared to asingle-drop fluorescence detection method can be increased by a factorof a/b for a given sensitivity, where a is the number of droplets thatcan be imaged within a given field-of-view, and b is the ratio of thefluorescence sensitivity of a single-drop fluorescence detector comparedto that of the multiple drop fluorescence detector. Furthermore, unlikesingle-drop fluorescent detection method where the drops are flowedthrough a detection volume so that their residence time in the detectionvolume, and hence the signal integration time and sensitivity, islimited, the residence time of the droplet in the field-of-view can beunlimited, thereby allowing sensitivity as high as the single-moleculelimit.

Beads

The device of the present invention also comprises the use of beads andmethods for analyzing and sorting beads (i.e, bead reader device). Thedevice can read and either sort or not sort droplets containing one ormore of a set of two or more beads. Each bead can be differentiated fromeach other bead within a set. Beads can be separated by several tagsincluding, but not limited to, quantum dyes, fluorescent dyes, ratios offluorescent dyes, radioactivity, radio-tags, etc. For example, a set ofbeads containing a ratio of two dyes in discrete amounts with anapparatus for detecting and differentiating beads containing onediscrete ratio from the other beads in this set having a different ratioof the two dyes. The microfluidic device can include paramagnetic beads.The paramagnetic beads can introduce and remove chemical components fromdroplets using droplet coalescence and breakup events. The paramagneticbeads can also be used for sorting droplets.

The present invention provides methods of screening molecular librarieson beads through limited-dilusion-loading and then chemical or opticalrelease inside of droplets. Provided are methods for chemical synthesison a bead and releasing said chemical attached to the bead using areleasing means (chemical, UV light, heat, etc) within a droplet, andthen combining a second droplet to the first droplet for furthermanipulation. For example, tea-bag synthesis of chemicals on a beadsimultaneously with a means for identifying said bead (using, forexample, a mass spec tag). Using the resulting mixed-chemistry beads ina droplet within a fluid flow, and exposing the beads to UV light torelease the chemical synthesized from the bead into the dropletenvironment. Combining the droplet containing the released chemical witha droplet containing a cell, and performing a cell-based assay. Sortingdroplets having the desired characteristics (for example, turn on of areporter gene), and then analyzing the sorted beads using massspectroscopy.

The device of the present invention can comprise column separation priorto bead sorting. A device containing a channel loaded with a separatingmeans for chromatographically sorting the sample prior to dropletformation. Such separating means could include size, charge,hydrophobicity, atomic mass, etc. The separating can be done isocraticor by use of a means for generating a gradient chemically, (for exampleusing salt or hydrophobicity), electrically, by pressure, or etc. Forexample, a channel is preloaded with Sepharose size exclusion media. Asample is loaded at one end, and the droplets are formed at an opposingend. The sample separates by size prior to becoming incorporated withina droplet.

Sorting Module

The microfluidic device of the present invention can further include oneor more sorting modules. A “sorting module” is a junction of a channelwhere the flow of molecules, cells, small molecules or particles canchange direction to enter one or more other channels, e.g., a branchchannel for delivery to an outlet module (i.e., collection or wastemodule), depending on a signal received in connection with anexamination in the detection module. Typically, a sorting module ismonitored and/or under the control of a detection module, and thereforea sorting module may “correspond” to such detection module. The sortingregion is in communication with and is influenced by one or more sortingapparatuses. A sorting apparatus comprises techniques or controlsystems, e.g., dielectric, electric, electro-osmotic, (micro-) valve,etc. A control system can employ a variety of sorting techniques tochange or direct the flow of molecules, cells, small molecules orparticles into a predetermined branch channel. A “branch channel” is achannel which is in communication with a sorting region and a mainchannel. The main channel can communicate with two or more branchchannels at the sorting module or “branch point”, forming, for example,a T-shape or a Y-shape. Other shapes and channel geometries may be usedas desired. Typically, a branch channel receives molecules, cells, smallmolecules or particles depending on the molecule, cells, small moleculesor particles characteristic of interest as detected by the detectionmodule and sorted at the sorting module. A branch channel can have anoutlet module and/or terminate with a well or reservoir to allowcollection or disposal (collection module or waste module, respectively)of the molecules, cells, small molecules or particles. Alternatively, abranch channel may be in communication with other channels to permitadditional sorting.

The device of the present invention can further include one or moreoutlet modules. An “outlet module” is an area of the device thatcollects or dispenses molecules, cells, small molecules or particlesafter coalescence, detection and/or sorting. The outlet module caninclude a collection module and/or a waste module. The collection modulecan be connected to a means for storing a sample. The collection modulecan be a well or reservoir for collecting and containing dropletsdetected to have a specific predetermined characteristic in thedetection module. The collection module can be temperature controlled.The waste module can be connected to a means for discarding a sample.The waste module can be a well or reservoir for collecting andcontaining droplets detected to not have a specific predeterminedcharacteristic in the detection module. The outlet module is downstreamfrom a sorting module, if present, or downstream from the detectionmodel if a sorting module is not present. The outlet module may containbranch channels or outlet channels for connection to a collection moduleor waste module. A device can contain more than one outlet module.

A characteristic of a fluidic droplet may be sensed and/or determined insome fashion, for example, as described herein (e.g., fluorescence ofthe fluidic droplet may be determined), and, in response, an electricfield may be applied or removed from the fluidic droplet to direct thefluidic droplet to a particular region (e.g. a channel). A fluidicdroplet is preferably sorted or steered by inducing a dipole in theuncharged fluidic droplet (which may be initially charged or uncharged),and sorting or steering the droplet using an applied electric field. Theelectric field may be an AC field, a DC field, etc. For example, withreference to FIG. 20A, a channel 540, containing fluidic droplet 530 andliquid 535, divides into channel 542 and 544. Fluidic droplet 530 isuncharged. Electrode 526 is positioned near channel 542, while electrode527 is positioned near channel 544. Electrode 528 is positioned near thejunction of channels 540, 542, and 544. In FIGS. 20C and 20D, a dipoleis induced in the fluidic droplet using electrodes 526, 527, and/or 528.In FIG. 20C, a dipole is induced in droplet 530 by applying an electricfield 525 to the droplet using electrodes 527 and 528. Due to thestrength of the electric field, the droplet is strongly attracted to theright, into channel 544. Similarly, in FIG. 20D, a dipole is induced indroplet 530 by applying an electric field 525 to the droplet usingelectrodes 526 and 528, causing the droplet to be attracted into channel542. Thus, by applying the proper electric field, droplet 530 can bedirected to either channel 542 or 544 as desired.

The present invention also provides improvements in the efficiency,accuracy, and reliability of the preferred dielectric droplet sortingtechnique described above. The single sided dielectric sorting relies ona combination of flow imbalance between the two exhaust legs and aswitchable electric field to selectively sort out droplets of interestfrom the main sample stream. Sorting decisions are made based on someform of real time measurement of the droplet and its contents. FIGS. 21and 22 depict many of the various possible fluid and electrodegeometries possible for single sided dielectric sorting. FIG. 21, PanelsA-D show possible flow channel geometries that can be used in anasymmetric sorting application. Panel F, illustrates the use of abarrier, for example, a barrier where no fluid flow passes on its leftside. Note these designs are only conceptual representation of the fluidchannels, and actual designs may differ in absolute and relativedimensions as determined by one of ordinary skill in the art.

FIG. 22 shows the possible electrode geometries used in an asymmetricsorting application. Panel A shows the use of sharp tipped electrodes.Panel B shows broad tipped electrodes to increase the interaction timebetween the droplets and the electric field (the tips could be many dropdiameters long). Panel C shows electrodes straddling the collectionline. Panel D shows electrodes on opposite sides of the main channel.Panel E shows an Asymmetric Electrode Pair (the asymmetry may be presenton any of the other electrode pair layouts as well). Note these designsare only conceptual representation of the electrodes, and actual designsmay differ in absolute dimensions and electrode shape as determined byone of ordinary skill in the art. Although the fluid channel geometry isdrawn as a “Y” junction, any of the channel geometries shown in FIG. 21could be substituted in these drawings.

Typically, the flow rate of the collection leg is set to a value justbelow the level required to begin pulling droplets into the collectionline (indicated as 40% in the figures, although the actual value may bediffer from this and is dependent on the actual fluidic and electrodegeometry, total flow, as well as droplet size and composition).

As an alternative design strategy, the collection leg can be operated ata flow rate at which the droplets would normally flow down the Sortcollect line (i.e. change the flow splits shown in the diagrams from 40%collect/60% waste to 60% collect/40% waste), and keep the electric fieldenergized until a droplet of interest is detected. At that time, thefield would be briefly turned off, and the droplet would be pulled downthe collection leg based on fluidic forces instead of electrical forces.

Alternately, a fluidic droplet may be directed by creating an electriccharge (e.g., as previously described) on the droplet, and steering thedroplet using an applied electric field, which may be an AC field, a DCfield, etc. As an example, an electric field maybe selectively appliedand removed (or a different electric field may be applied) as needed todirect the fluidic droplet to a particular region. The electric fieldmay be selectively applied and removed as needed, in some embodiments,without substantially altering the flow of the liquid containing thefluidic droplet. For example, a liquid may flow on a substantiallysteady-state basis (i.e., the average flowrate of the liquid containingthe fluidic droplet deviates by less than 20% or less than 15% of thesteady-state flow or the expected value of the flow of liquid withrespect to time, and in some cases, the average flowrate may deviateless than 10% or less than 5%) or other predetermined basis through afluidic system of the invention (e.g., through a channel or amicrochannel), and fluidic droplets contained within the liquid may bedirected to various regions, e.g., using an electric field, withoutsubstantially altering the flow of the liquid through the fluidicsystem.

In other embodiments, however, the fluidic droplets may be screened orsorted within a fluidic system of the invention by altering the flow ofthe liquid containing the droplets. For instance, in one set ofembodiments, a fluidic droplet may be steered or sorted by directing theliquid surrounding the fluidic droplet into a first channel, a secondchannel, etc.

In another set of embodiments, pressure within a fluidic system, forexample, within different channels or within different portions of achannel, can be controlled to direct the flow of fluidic droplets. Forexample, a droplet can be directed toward a channel junction includingmultiple options for further direction of flow (e.g., directed toward abranch, or fork, in a channel defining optional downstream flowchannels). Pressure within one or more of the optional downstream flowchannels can be controlled to direct the droplet selectively into one ofthe channels, and changes in pressure can be effected on the order ofthe time required for successive droplets to reach the junction, suchthat the downstream flow path of each successive droplet can beindependently controlled. In one arrangement, the expansion and/orcontraction of liquid reservoirs may be used to steer or sort a fluidicdroplet into a channel, e.g., by causing directed movement of the liquidcontaining the fluidic droplet. The liquid reservoirs may be positionedsuch that, when activated, the movement of liquid caused by theactivated reservoirs causes the liquid to flow in a preferred direction,carrying the fluidic droplet in that preferred direction. For instance,the expansion of a liquid reservoir may cause a flow of liquid towardsthe reservoir, while the contraction of a liquid reservoir may cause aflow of liquid away from the reservoir. In some cases, the expansionand/or contraction of the liquid reservoir may be combined with otherflow-controlling devices and methods, e.g., as described herein. Nonlimiting examples of devices able to cause the expansion and/orcontraction of a liquid reservoir include pistons and piezoelectriccomponents. In some cases, piezoelectric components may be particularlyuseful due to their relatively rapid response times, e.g., in responseto an electrical signal.

In some embodiments, the fluidic droplets may be sorted into more thantwo channels. Alternately, a fluidic droplet may be sorted and/or splitinto two or more separate droplets, for example, depending on theparticular application. Any of the above-described techniques may beused to spilt and/or sort droplets. As a non-limiting example, byapplying (or removing) a first electric field to a device (or a portionthereof), a fluidic droplet may be directed to a first region orchannel; by applying (or removing) a second electric field to the device(or a portion thereof), the droplet may be directed to a second regionor channel; by applying a third electric field to the device (or aportion thereof), the droplet may be directed to a third region orchannel; etc., where the electric fields may differ in some way, forexample, in intensity, direction, frequency, duration, etc. In a seriesof droplets, each droplet may be independently sorted and/or split; forexample, some droplets may be directed to one location or another, whileother droplets may be split into multiple droplets directed to two ormore locations.

In some cases, high sorting speeds may be achievable using certainsystems and methods of the invention. For instance, at least about 1droplet per second may be determined and/or sorted in some cases, and inother cases, at least about 10 droplets per second, at least about 20droplets per second, at least about 30 droplets per second, at leastabout 100 droplets per second, at least about 200 droplets per second,at least about 300 droplets per second, at least about 500 droplets persecond, at least about 750 droplets per second, at least about 1000droplets per second, at least about 1500 droplets per second, at leastabout 2000 droplets per second, at least about 3000 droplets per second,at least about 5000 droplets per second, at least about 7500 dropletsper second, at least about 10,000 droplets per second, at least about15,000 droplets per second, at least about 20,000 droplets per second,at least about 30,000 droplets per second, at least about 50,000droplets per second, at least about 75,000 droplets per second, at leastabout 100,000 droplets per second, at least about 150,000 droplets persecond, at least about 200,000 droplets per second, at least about300,000 droplets per second, at least about 500,000 droplets per second,at least about 750,000 droplets per second, at least about 1,000,000droplets per second may be determined and/or sorted in such a fashion.

Multiple Measurement Sorting

In some embodiments, it may be useful to sort droplets based on twodifferent measurements. For example, one might want to sort based on theratio of two signals, sum of two signals, or difference between twosignals. Specifically, this would be useful for cases when one wouldlike to optimize an enzyme so that it work one substrate, but notanother, or so that it works on two substrates. This is not easy to dousing multiple rounds of selection on populations of droplets. Toovercome this shortcoming of current sorting technology, the presentinvention provides a device comprising multiple channels with theappropriate geometry to split droplets, perform different experiments onthe two daughter droplets and then reorder so that they pass sequentialthrough the detector. The sums, ratios or differences in the two signalscan then be calculated before the droplets enter the sortingbifurcation. An indicator dye or equivalent material may be added to oneor both droplets to indicate when each droplet enters and leaves thelaser. A representative sketch is shown in FIG. 23.

Sample Recovery

The present invention proposes methods for recovering aqueous phasecomponents from aqueous emulsions that have been collected on amicrofluidic device in a minimum number of steps and in a gentle mannerso as to minimize potential damage to cell viability.

In one aspect, a stable aqueous sample droplet emulsion containingaqueous phase components in a continuous phase carrier fluid is allowedto cream to the top of the continuous phase carrier oil. By way ofnonlimiting example, the continuous phase carrier fluid can includeperfluorocarbon oil that can have one or more stabilizing surfactants.The aqueous emulsion rises to the top or separates from the continuousphase carrier fluid by virtue of the density of the continuous phasefluid being greater than that of the aqueous phase emulsion. Forexample, the perfluorocarbon oil used in one embodiment of the device is1.8, compared to the density of the aqueous emulsion, which is 1.0.

The creamed emulsion is then placed onto a second continuous phasecarrier fluid which contains a de-stabilizing surfactant, such as aperfluorinated alcohol (e.g., 1H, 1H, 2H,2H-Perfluoro-1-octanol). Thesecond continuous phase carrier fluid can also be a perfluorocarbon oil.Upon mixing, the aqueous emulsion begins to coalesce, and coalescence iscompleted by brief centrifugation at low speed (e.g., 1 minute at 2000rpm in a microcentrifuge). The coalesced aqueous phase can now beremoved (cells can be placed in an appropriate environment for furtheranalysis).

Additional destabilizing surfactants and/or oil combinations can beidentified or synthesized to be useful with this invention.

Mixing Module

The microfluidic device of the present invention can further include oneor more mixing modules. Although coalescence of one or more droplets inone or more coalescence modules can be sufficient to mix the contents ofthe coalesced droplets (e.g., through rotating vortexes existing withinthe droplet), it should be noted that when two droplets fuse orcoalesce, perfect mixing within the droplet does not instantaneouslyoccur. Instead, for example, the coalesced droplet may initially beformed of a first fluid region (from the first droplet) and a secondfluid region (from the second droplet). Thus, in some cases, the fluidregions may remain as separate regions, for example, due to internal“counter-revolutionary” flow within the fluidic droplet, thus resultingin a non-uniform fluidic droplet. A “mixing module” can comprisefeatures for shaking or otherwise manipulate droplets so as to mix theircontents. The mixing module is preferably downstream from the coalescingmodule and upstream from the detection module. The mixing module caninclude, but is not limited to, the use of channel geometries, acousticactuators, metal alloy component electrodes or electrically conductivepatterned electrodes to mix the contents of droplets and to reducemixing times for fluids combined into a single droplet in themicrofluidic device. For example, the fluidic droplet may be passedthrough one or more channels or other systems which cause the droplet tochange its velocity and/or direction of movement. The change ofdirection may alter convection patterns within the droplet, causing thefluids to be at least partially mixed. Combinations are also possible.

For acoustic manipulation, the frequency of the acoustic wave should befine tuned so as not to cause any damage to the cells. The biologicaleffects of acoustic mixing have been well studied (e.g., in the ink-jetindustry) and many published literatures also showed that piezoelectricmicrofluidic device can deliver intact biological payloads such as livemicroorganisms and DNA. In an example, the design of the acousticresonant uses a Piezoelectric bimorph flat plate located on the side ofthe carved resonant in the PDMS slab. The piezoelectric driving waveformis carefully optimized to select the critical frequencies that canseparate cells in fluids. There are five parameters to optimize beyondthe frequency parameter. Lab electronics is used to optimize thepiezoelectric driving waveform. Afterwards, a low cost circuit can bedesigned to generate only the optimized waveform in a preferredmicrofluidic device.

Other examples of fluidic mixing in droplets are described WO2004/091763, incorporated herein by reference.

Delay Module

The microfluidic device of the present invention can further include oneor more delay modules. The “delay module” can be a delay line. Theoperation of a microfluidics device where a reaction within a droplet isallowed to occur for a non-trivial length of time requires a delay lineto increase the residence time within the device. For reactionsdemanding extensive residence time, longer or larger delay lines arerequired. Accordingly, the invention provides methods to increaseresidence times within microfluidic devices.

The delay module is in fluid communication with the main channel or itcan be an elongated portion of the main channel itself. The delay modulecan be located downstream of the coalescence module and upstream of thedetection module. The delay module can be a serpentine channel or abuoyant hourglass. The delay module can further comprise heating andcooling regions. The heating and cooling regions can be used forperforming on-chip, flow-through PCR as further described herein.

The channel dimensions and configurations can be designed to accommodatethe required residence time with minimum pressure drops across thedevice. For example, to accommodate very long delay lines within themicrofluidic device, the device can comprise a multilayered PDMS slabwhich is composed of several patterned PDMS slabs.

The channel dimensions can also be designed so as to allow for requiredflow, residence time and pressure drop. Some channels may be required tobe very large in width and height. In order to avoid collapse of thechannels, the device includes support posts within the channel design.In order to reduce dead volume behind posts and further improve dropletstability, the support posts are designed to optimize a streamlined flowwithin the channel. These designs can include curved features as opposedto sharp edges.

To allow for longer period of device operation, delay lines can also beextended to the outside of the chip. The off-chip delay lines can betubes within micron-sized internal diameter.

In order to allow more efficient use of available space and fasteroperation, in methods where droplets are charged, after charging,asymmetric splitting of oil and drops can be accommodated by siphoningoff oil from channels after droplets are charged.

The delay lines can be in the form of a tower (i.e., a structure whichis vertical with respect to the ambient gravitational field) as to allowbuoyant forces to assist controlled droplet transport. Known delay linesinvolve transporting droplets by emulsifying them in a carrier fluidflowing in a channel and/or tube. Because the velocity profile of thecarrier fluid through the cross-section of the channel and/or tube isnot uniform, the velocity distribution of the droplets will not benarrow, which causes the delay time distribution of the droplets to notbe narrow (i.e., some droplets will be delayed more or less thanothers).

The devices of the present invention can also include buoyancy-assistedmicrofluidic delay lines. In buoyancy-assisted microfluidic delay lines,buoyant forces act on droplets emulsified in a fluid in one or moretowers. This can include allowing the tower to fill for the desireddelay time, and then releasing the droplets. The tower can or can notcontinue to fill and release droplets as needed. In this example, onemay desire to have a cylindrical tower section that is capped by apyramidal funnel section. The tower can effectively functions as anhourglass. Droplets that have a density less than their carrier fluidare fed into the base of the tower, buoyantly rise to the top of thetower with a substantially uniform velocity distribution, and arefunneled into a functional component of the microfluidic device (such asa y-branch). Carrier fluid is exhausted at the base of the tower at thesame rate as it is introduced at the apex so that the net flow ofcarrier fluid through the delay line is zero. The tower and funnelsections can have any cross-sectional shape, such as circular,elliptical, or polygonal. The microfluidic device can include a towerwith adjustable length.

The device can also include a switching network of twenty towers toguarantee a delay time dispersion of 5% (because 1/20=0.05). Thecapacity of each tower is 0.05*T, where T is the delay time. The conceptincludes, for example: (a) upon device start-up, filling the first towerfor 0.05*T, but stop-cock its exhaust, and also have the other nineteentowers closed; (b) after 0.05*T, closing the first tower and filling thesecond between 0.05*T and 0.10*T; (c) repeating step (b) for theremaining eighteen towers; (d) at time T, allowing the first tower toexhaust; (e) at time 1.05*T, stop-cocking the exhaust of the firsttower, allowing the second tower to exhaust, and allowing the firsttower to fill; (f) at time 1.10*T, stop-cocking the exhaust of thesecond tower, allowing the third tower to exhaust, closing the firsttower, and allowing the second tower to fill, and (g) repeating step (f)ad infinitum. More than twenty towers may provide an even tightercontrol over the width of the delay time dispersion. This scheme mayrequire a valve network. This network of towers can be outside themicrofluidic device.

The delay module can also include channels (e.g. the main channel) whichhas an altered geometry which permits the “parking” (e.g., slowing orstopping) of droplets within the microfluidic device.

In the methods provided herein, droplets are able to be parked in wellsor channels at predefined locations. This can be done by creatingdiscrete well-like indentions in the channel whereby a droplet ‘falls’into the well and remains there as the fluid flows over it, or by usinga technique entitled ‘by-pass pots’ whereby a droplet is used to block asmall outlet in a well, thereby causing the flow to by-pass thatdroplet-containing well.

The instant invention is to use either of these techniques or anyrelated technique, for example just stopping the drops in a channel, toposition droplets at either random or predefined places within amicrofluidics device. These random or predefined locations can then bequeried at a later time-point for a reaction to have occurred, or forremoval of the droplets using another means such as resuspensionfollowed by aspiration.

In one example, a rolling circle amplification reaction is initiated indroplets, the droplets are then parked within the chip, and theamplification reaction allowed to proceed for a set period of time priorto stopping the reaction through the use of heat. The parked dropletsare then dried in situ and the covering of the chip disassembled fromthe chip. One or a set of needle-like devices that are able to be linedup with the droplet parking space are then placed adjacent to or on topof the dried droplets and a liquid solution used to resuspend thematerial in the dried droplet that has been deposited into the chip, forfurther downstream processing.

In another example, to avoid possible diffusion of reactant contentsfrom a first and a second set of reactions in droplets, the firstreactions are created in 10 μm droplets, the droplets are dried within achannel parking space or by-pass pot which is able to hold a droplet ofsize larger than 10 μm, and the droplets are dried in situ. A second setof droplets that are larger than 10 μm are then allowed to proceed downsaid channel and when caught in said parking space or by-pass pot areable to resuspend the material from the first droplets that are driedalong the walls of the first parking space or by-pass pot. In doing so,the second droplet is slightly larger than the first and that ensuresthat the material along the walls is ‘captured’ by the second droplet,and not allowed to diffuse away from the first droplet wall bydiffusion. By doing so, use of surfactants becomes optional in eitherthe first or second droplet formulations.

The instant invention also provides the following devices and methodsfor use in practicing the methods described herein. The PDMS substratewhich comprises a portion of the microfluidic device can be covered orcoated with an adhesive tape or strip that can removed by peeling. ThePDMS substrate can also be bonded by an ultra thin silica that can bepierced by a set of needles. The silica may be spin coated orelectro-plated onto a thin backing. Droplets can be dried onto a pieceof paper such that can be detected by a second device to determine theNcode within the droplet and to determine whether an amplificationreaction has occurred within the droplet. A plate read comprising driedand undried spots using either an optical array device, such as found inhigh-end cameras or fiber, optic device is also contemplated. DryNitrogen can be utilized to dry the spots by either flowing it throughthe channel or placing the device into a dry-N2 chamber. Channels can befilled with dried nitrogen or salt run underneath or adjacent to theparking space channels to allow chemical or physical-type gradients tobe set up in the chip. The channel walls can be coated with Steptavidinand the produced reactants, for example, DNA biotinylated so that itadheres in situ. Porous beads deposited into the wells can be used incombination with solutions without oils to wash the beads by flow,followed by re-depositing droplets with surfactants to recoat the beads.The wells within the substrate can be filled with many small beads byloading small beads into droplets, storing the droplets into individualwells containing apertures that are slightly smaller than the beads,breaking the droplets by drying or flow of aqueous solutions with orwithout surfactants into the channels and past the beads, and thenre-encapsulating the beads in situ. A set of electrodes within oradjacent to the microfluidic substrate can be used to fuse two dropletsin a storage/holding space. The electrodes may be perpendicular to theplane of the channels and either the electrodes or channels moved so asto allow droplet fusions to occur.

UV-Release Module

The microfluidic device of the present invention can further include oneor more UV-release modules. The “UV-release module” is in fluidcommunication with the main channel. The UV release module is locateddownstream of the inlet module and upstream of the coalescence module.The UV-module can be a used in bead assays. Compounds from encapsulatedbeads can be cleaved in a UV-releasing module using UV light.Photolabile linkers can be broken down on demand after a single bead hasbeen encapsulated thus releasing multiple copies of a single compoundinto solution. In the cell based assay disclosed herein the chemicalcompound assayed is desired to be in solution in order to penetrate thecell membrane. Furthermore, to ensure compartmentalization of a single,compound with a cell the cleavage of the compound from the solid supportcan only be done after the bead has been encapsulated. Photocleavablelinkers can be utilized to cleave the compounds of the bead after dropformation by passing the drop through a UV-release module (i.e., laserof the appropriate wavelength).

The present invention also provides methods for chemical synthesis on abead and releasing said chemical attached to the bead using a releasingmeans (chemical, UV light, heat, etc.) within a droplet, and thencombining a second droplet to the first droplet for furthermanipulation. Preferably, the releasing means is a UV-module. Forexample, tea-bag synthesis of chemicals on a bead simultaneously with ameans for identifying said bead (using, for example, a mass spec tag).Using the resulting mixed-chemistry beads in a droplet within a fluidflow, and exposing the beads to UV light to release the chemicalsynthesized from the bead into the droplet environment. Combining thedroplet containing the released chemical with a droplet containing acell, and performing a cell-based assay. Sorting droplets having thedesired characteristics (for example, turn on of a reporter gene), andthen analyzing the sorted beads using mass spectroscopy.

Kits

As a matter of convenience, predetermined amounts of the reagents,compound libraries, and/or emulsions described herein and employed inthe present invention can be optionally provided in a kit in packagedcombination to facilitate the application of the various assays andmethods described herein. Such kits also typically include instructionsfor carrying out the subject assay, and may optionally include the fluidreceptacle, e.g., the cuvette, multiwell plate, microfluidic device,etc. in which the reaction is to be carried out.

Typically, reagents included within the kit are uniquely labeledemulsions containing tissues, cells, particles, proteins, antibodies,amino acids, nucleotides, small molecules, substrates, and/orpharmaceuticals. These reagents may be provided in pre-measuredcontainer (e.g., vials or ampoules) which are co-packaged in a singlebox, pouch or the like that is ready for use. The container holding thereagents can be configured so as to readily attach to the fluidreceptacle of the device in which the reaction is to be carried out(e.g., the inlet module of the microfluidic device as described herein).In one embodiment, the kit can include an RNAi kit. In anotherembodiment, the kit can include a chemical synthesis kit. It will beappreciated by persons of ordinary skill in the art that theseembodiments are merely illustrative and that other kits are also withinthe scope of the present invention.

Methods

The microfluidic device of the present invention can be utilized toconduct numerous chemical and biological assays, including but notlimited to, creating emulsion libraries, flow cytometry, geneamplification, isothermal gene amplification, DNA sequencing, SNPanalysis, drug screening, RNAi analysis, karyotyping, creating microbialstrains with improved biomass conversion, moving cells using opticaltweezer/cell trapping, transformation of cells by electroporation, μTAS,and DNA hybridization.

Definitions

The terms used in this Specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the devices and methods of theinvention and how to make and use them. It will be appreciated that thesame thing can typically be described in more than one way.Consequently, alternative language and synonyms may be used for any oneor more of the terms discussed herein. Synonyms for certain terms areprovided. However, a recital of one or more synonyms does not excludethe use of other synonyms, nor is any special significance to be placedupon whether or not a term is elaborated or discussed herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference. In the case of conflict,the present specification, including definitions, will control. Inaddition, the materials, methods, and examples are illustrative only andare not intended to be limiting.

The invention is also described by means of particular examples.However, the use of such examples anywhere in the specification,including examples of any terms discussed herein, is illustrative onlyand in no way limits the scope and meaning of the invention or of anyexemplified term. Likewise, the invention is not limited to anyparticular preferred embodiments described herein. Indeed, manymodifications and variations of the invention will be apparent to thoseskilled in the art upon reading this specification and can be madewithout departing from its spirit and scope. The invention is thereforeto be limited only by the terms of the appended claims along with thefull scope of equivalents to which the claims are entitled.

As used herein, “about” or “approximately” shall generally mean within20 percent, preferably within 10 percent, and more preferably within 5percent of a given value or range.

The term “molecule” means any distinct or distinguishable structuralunit of matter comprising one or more atoms, and includes for examplepolypeptides and polynucleotides.

The term “polymer” means any substance or compound that is composed oftwo or more building blocks (‘mers’) that are repetitively linked toeach other. For example, a “dimer” is a compound in which two buildingblocks have been joined together.

The term “polynucleotide” as used herein refers to a polymeric moleculehaving a backbone that supports bases capable of hydrogen bonding totypical polynucleotides, where the polymer backbone presents the basesin a manner to permit such hydrogen bonding in a sequence specificfashion between the polymeric molecule and a typical polynucleotide(e.g., single-stranded DNA). Such bases are typically inosine,adenosine, guanosine, cytosine, uracil and thymidine. Polymericmolecules include double and single stranded RNA and DNA, and backbonemodifications thereof, for example, methylphosphonate linkages.

Thus, a “polynucleotide” or “nucleotide sequence” is a series ofnucleotide bases (also called “nucleotides”) generally in DNA and RNA,and means any chain of two or more nucleotides. A nucleotide sequencetypically carries genetic information, including the information used bycellular machinery to make proteins and enzymes. These terms includedouble or single stranded genomic and cDNA, RNA, any synthetic andgenetically manipulated polynucleotide, and both sense and anti-sensepolynucleotide (although only sense stands are being representedherein). This includes single- and double-stranded molecules, i.e.,DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids”(PNA) formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases, for examplethio-uracil, thio-guanine and fluoro-uracil.

The polynucleotides herein may be flanked by natural regulatorysequences, or may be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenylation sequences, introns, 5′- and 3′-non-coding regions, andthe like. The nucleic acids may also be modified by many means known inthe art. Non-limiting examples of such modifications includemethylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, and internucleotide modificationssuch as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) andwith charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.). Polynucleotides may contain one or more additional covalentlylinked moieties, such as, for example, proteins (e.g., nucleases,toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators(e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactivemetals, iron, oxidative metals, etc.), and alkylators. Thepolynucleotides may be derivatized by formation of a methyl or ethylphosphotriester or an alkyl phosphoramidate linkage. Furthermore, thepolynucleotides herein may also be modified with a label capable ofproviding a detectable signal, either directly or indirectly. Exemplarylabels include radioisotopes, fluorescent molecules, biotin, and thelike.

The term “dielectrophoretic force gradient” means a dielectrophoreticforce is exerted on an object in an electric field provided that theobject has a different dielectric constant than the surrounding media.This force can either pull the object into the region of larger field orpush it out of the region of larger field. The force is attractive orrepulsive depending respectively on whether the object or thesurrounding media has the larger dielectric constant.

“DNA” (deoxyribonucleic acid) means any chain or sequence of thechemical building blocks adenine (A), guanine (G), cytosine (C) andthymine (T), called nucleotide bases, that are linked together on adeoxyribose sugar backbone. DNA can have one strand of nucleotide bases,or two complimentary strands which may form a double helix structure.“RNA” (ribonucleic acid) means any chain or sequence of the chemicalbuilding blocks adenine (A), guanine (G), cytosine (C) and uracil (U),called nucleotide bases, that are linked together on a ribose sugarbackbone. RNA typically has one strand of nucleotide bases.

A “polypeptide” (one or more peptides) is a chain of chemical buildingblocks called amino acids that are linked together by chemical bondscalled peptide bonds. A “protein” is a polypeptide produced by a livingorganism. A protein or polypeptide may be “native” or “wild-type”,meaning that it occurs in nature; or it may be a “mutant”, “variant” or“modified”, meaning that it has been made, altered, derived, or is insome way different or changed from a native protein, or from anothermutant.

An “enzyme” is a polypeptide molecule, usually a protein produced by aliving organism, that catalyzes chemical reactions of other substances.The enzyme is not itself altered or destroyed upon completion of thereaction, and can therefore be used repeatedly to catalyze reactions. A“substrate” refers to any substance upon which an enzyme acts.

As used herein, “particles” means any substance that may be encapsulatedwithin a droplet for analysis, reaction, sorting, or any operationaccording to the invention. Particles are not only objects such asmicroscopic beads (e.g., chromatographic and fluorescent beads), latex,glass, silica or paramagnetic beads, but also includes otherencapsulating porous and/or biomaterials such as liposomes, vesicles andother emulsions. Beads ranging in size from 0.1 micron to 1 mm can beused in the devices and methods of the invention and are thereforeencompassed with the term “particle” as used herein. The term particlealso encompasses biological cells, as well as beads and othermicroscopic objects of similar size (e.g., from about 0.1 to 120microns, and typically from about 1 to 50 microns) or smaller (e.g.,from about 0.1 to 150 nm). The devices and methods of the invention arealso directed to sorting and/or analyzing molecules of any kind,including polynucleotides, polypeptides and proteins (including enzymes)and their substrates and small molecules (organic or inorganic). Thus,the term particle further encompasses these materials.

The particles (including, e.g., cells and molecules) are sorted and/oranalyzed by encapsulating the particles into individual droplets (e.g.,droplets of aqueous solution in oil), and these droplets are thensorted, combined and/or analyzed in a microfabricated device.Accordingly, the term “droplet” generally includes anything that is orcan be contained within a droplet.

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart.

As used herein, “cell” means any cell or cells, as well as viruses orany other particles having a microscopic size, e.g. a size that issimilar to or smaller than that of a biological cell, and includes anyprokaryotic or eukaryotic cell, e.g., bacteria, fungi, plant and animalcells. Cells are typically spherical, but can also be elongated,flattened, deformable and asymmetrical, i.e., non-spherical. The size ordiameter of a cell typically ranges from about 0.1 to 120 microns, andtypically is from about 1 to 50 microns. A cell may be living or dead.Since the microfabricated device of the invention is directed to sortingmaterials having a size similar to a biological cell (e.g. about 0.1 to120 microns) or smaller (e.g., about 0.1 to 150 nm) any material havinga size similar to or smaller than a biological cell can be characterizedand sorted using the microfabricated device of the invention. Thus, theterm cell shall further include microscopic beads (such aschromatographic and fluorescent beads), liposomes, emulsions, or anyother encapsulating biomaterials and porous materials. Non-limitingexamples include latex, glass, orparamagnetic beads; and vesicles suchas emulsions and liposomes, and other porous materials such as silicabeads. Beads ranging in size from 0.1 micron to 1 mm can also be used,for example in sorting a library of compounds produced by combinatorialchemistry. As used herein, a cell may be charged or uncharged. Forexample, charged beads may be used to facilitate flow or detection, oras a reporter. Biological cells, living or dead, may be charged forexample by using a surfactant, such as SDS (sodium dodecyl sulfate). Theterm cell further encompasses “virions”, whether or not virions areexpressly mentioned.

A “virion”, “virus particle” is the complete particle of a virus.Viruses typically comprise a nucleic acid core (comprising DNA or RNA)and, in certain viruses, a protein coat or “capsid” Certain viruses mayhave an outer protein covering called an “envelope”. A virion may beeither living (i.e., “viable”) or dead (i.e., “non-viable”). A living or“viable” virus is one capable of infecting a living cell. Viruses aregenerally smaller than biological cells and typically range in size fromabout 20-25 nm diameter or less (parvoviridae, picornoviridae) toapproximately 200-450 nm (poxviridae). However, some filamentous virusesmay reach lengths of 2000 nm (closterviruses) and are therefore largerthan some bacterial cells. Since the microfabricated device of theinvention is particularly suited for sorting materials having a sizesimilar to a virus (i.e., about 0.1 to 150 nm), any material having asize similar to a virion can be characterized and sorted using themicrofabricated device of the invention. Non-limiting examples includelatex, glass or paramagnetic beads; vesicles such as emulsions andliposomes; and other porous materials such as silica beads. Beadsranging in size from 0.1 to 150 nm can also be used, for example, insorting a library of compounds produced by combinatorial chemistry. Asused herein, a virion may be charged or uncharged. For example, chargedbeads may be used to facilitate flow or detection, or as a reporter.Biological viruses, whether viable or non-viable, may be charged, forexample, by using a surfactant, such as SDS.

A “reporter” is any molecule, or a portion thereof, that is detectable,or measurable, for example, by optical detection. In addition, thereporter associates with a molecule, cell or virion or with a particularmarker or characteristic of the molecule, cell or virion, or is itselfdetectable to permit identification of the molecule, cell or virion's,or the presence or absence of a characteristic of the molecule, cell orvirion. In the case of molecules such as polynucleotides suchcharacteristics include size, molecular weight, the presence or absenceof particular constituents or moieties (such as particular nucleotidesequences or restrictions sites). In the case of cells, characteristicswhich may be marked by a reporter includes antibodies, proteins andsugar moieties, receptors, polynucleotides, and fragments thereof. Theterm “label” can be used interchangeably with “reporter”. The reporteris typically a dye, fluorescent, ultraviolet, or chemiluminescent agent,chromophore, or radio-label, any of which may be detected with orwithout some kind of stimulatory event, e.g., fluoresce with or withouta reagent. In one embodiment, the reporter is a protein that isoptically detectable without a device, e.g. a laser, to stimulate thereporter, such as horseradish peroxidase (HRP). A protein reporter canbe expressed in the cell that is to be detected, and such expression maybe indicative of the presence of the protein or it can indicate thepresence of another protein that may or may not be coexpressed with thereporter. A reporter may also include any substance on or in a cell thatcauses a detectable reaction, for example by acting as a startingmaterial, reactant or a catalyst for a reaction which produces adetectable product. Cells may be sorted, for example, based on thepresence of the substance, or on the ability of the cell to produce thedetectable product when the reporter substance is provided.

A “marker” is a characteristic of a molecule, cell or virion that isdetectable or is made detectable by a reporter, or which may becoexpressed with a reporter. For molecules, a marker can be particularconstituents or moieties, such as restrictions sites or particularnucleic acid sequences in the case of polynucleotides. For cells andvirions, characteristics may include a protein, including enzyme,receptor and ligand proteins, saccharrides, polynucleotides, andcombinations thereof, or any biological material associated with a cellor virion. The product of an enzymatic reaction may also be used as amarker. The marker may be directly or indirectly associated with thereporter or can itself be a reporter. Thus, a marker is generally adistinguishing feature of a molecule, cell or virion, and a reporter isgenerally an agent which directly or indirectly identifies or permitsmeasurement of a marker. These terms may, however, be usedinterchangeably.

The invention is further described below, by way of the followingexamples. The examples also illustrate useful methodology for practicingthe invention. These examples do not limit the claimed invention.

EXAMPLES Example 1

The present invention provides methods for preparing a library ofdroplet emulsions, where each of the droplets is of the same,predetermined size (monodisperse). Further, present invention providesmethods for deterministic lateral displacement for continuous particleseparation, which can occur within droplets on a microfluidic device.

Particles in solution are usually separated according to size byexclusion or hydrodynamic chromatography. In the former, a samplemixture is injected at one end of a tube packed with porous beads andthen washed through the tube. Particles smaller than the pore sizesenter the beads, which lengthen their migration path, and so they are onaverage eluted later than larger particles. Zones of particles of agiven size broaden, however, because particles in each zone take manydifferent paths leading to different retention times. This multipatheffect reduces the resolution of size-exclusion chromatography. Inhydrodynamic chromatography, a sample mixture is driven through acapillary by hydrodynamic flow, which has a parabolic flow profile.Large particles cannot intercept the low-velocity fluid near thecapillary wall, and thus on average move faster and become separatedfrom small particles. Multipath effects also limit the resolution ofhydrodynamic chromatography, because each migration path samplesdifferent velocities in the parabolic flow.

Recently, Huang et al. Science 304(5673):987-90, 2004 and Davis et alProc Natl Acad Sci USA. 103(40):14779-84, 2006 demonstrate a separationprocess that creates equivalent migration paths for each particle in amixture, thereby eliminating multipath zone broadening. They describe a‘lateral displacement’ means for separation of particles in solutionbased on particle size.

Lateral displacement means for sizing and separating droplets insolution (based on droplet size) can be utilized. The present inventionrelates to the generation of a microfluidic device consisting of raisedpillars in both columns and rows that are designed for lateraldiffusion. The pillars can be adjusted so as to be a means forseparating droplets of similar sizes from a fluid containing varioussized droplets.

In an example, a fluid containing oil, water and a surfactant is mixedso as to create a bulk emulsion. The bulk emulsion is injected intobeginning of a microfluidic lateral diffusion device and variousfractions are collected at the ending of the device at positionscorresponding to specific sizes. Advantages to this lateral diffusionseparation means would be the isolation of similarly-sized dropletsoff-line in a fast and facile manner. Bulk emulsions could besize-selected and then the resulting emulsions, if desired, combined tocreate sized libraries for re-introduction into a microfluidic device.In a further example, the lateral diffusion microfluidic devices couldbe rolled-up into a syringe or designed for parallel processing.

Recently, devices that exploit both techniques have been miniaturizedwith the use of microfabrication technology. Microfabricated deviceshave also been designed that inherently rely on diffusion forseparation. Particle mixtures are either repeatedly subject to spatiallyasymmetric potentials created by microelectrodes or driven througharrays of micrometer-scale asymmetric obstacles to exploit differencesin diffusion lengths. In all of the devices discussed so far, particlesin a given zone have many different migration paths, and diffusion isrequired for separation.

The present invention describes a separation process that createsequivalent migration paths for each particle in a mixture, therebyeliminating multipath zone broadening (FIG. 24). FIG. 24. (Panel A)Geometric parameters defining the obstacle matrix. A fluid flow isapplied in the vertical direction (orange arrow). (Panel B) Three fluidstreams (red, yellow, and blue) in a gap do not mix as they flow throughthe matrix. Lane 1 at the first obstacle row becomes lane 3 at thesecond row, lane 3 becomes lane 2 at the third row, and so on. Smallparticles following streamlines will thus stay in the same lane. (PanelC) A particle with a radius that is larger than lane 1 follows astreamline passing through the particle's center (black dot), movingtoward lane 1. The particle is physically displaced as it enters thenext gap. Black dotted lines mark the lanes.

The separation process uses laminar flow through a periodic array ofmicrometer-scale obstacles. Each row of obstacles is shiftedhorizontally with respect to the previous row by δλ, where λ is thecenter-to-center distance between the obstacles (FIG. 24). Forconvenience, let δλ/λ be 1/3. Fluid emerging from a gap between twoobstacles will encounter an obstacle in the next row and will bifurcateas it moves around the obstacle. Let the flow diverted to the left ofthe obstacle be δϕ, where ϕ is the total fluid flux going through thegap. If the fluid is confined to move straight down through the array, δmust equal δλ/λ. Let us then consider the flow through a gap to be madeup of three lanes, each of which by definition has a flux of ϕ/3.Because the Reynolds number is low (<10³ in micrometer-scaleenvironments) and flows are laminar, the streams in each lane do notcross or mix (FIG. 24B). Notably, as the lanes go through gaps, theirpositions relative to the gaps change. The lanes are represented in eachgap by 1, 2, and 3, from left to right, respectively. Lane 1 becomeslane 3 in the next gap, lane 2 becomes lane 1, and lane 3 becomes lane 2(FIG. 24). After three rows, the three lanes rejoin in their originalconfiguration.

Particles that are smaller than the lane width will follow thestreamlines. A particle starting in lane 1 will go through lane 3 (rightlane with respect to the gap) in the second row, lane 2 (middle lane) inthe third row, and back to lane 1 (left lane) in the fourth row (FIG.24B). In fact, particles starting from any of the three lanes will goback to the original lane assignment after three rows, so that netmigration is in the average flow direction. This motion is called the“zigzag mode.” In practice, particles can diffuse into an adjacent lane.However, the microscopic path for all lanes is equivalent, unlike themultiple paths particles take when moving through a column of porousbeads. In contrast to the smaller particles, a particle with a radiuslarger than the width of lane 1 at a gap will behave differently in thearray. This is because the center of the particle cannot “fit” into lane1 in a gap. As such a particle from lane 2 in one gap moves into thesubsequent gap, expecting to move through the gap in lane 1, theparticle will be “bumped” and its center will thus be displaced intolane 2 (FIG. 24C). The particle will then flow with the fluid in lane 2.This process is repeated every time a large particle approaches a row ofobstacles, so that the particle remains in lane 2 as it moves downthrough the array. This transport pattern is called the “displacementmode.” This is also applicable to electrophoresis by considering ionflows instead of fluid flows.

FIG. 25 shows High-resolution separation of fluorescent microsphereswith diameters of 0.80 um (green), 0.90 um (red), and 1.03 um (yellow),with a matrix of varying gap size. Whereas the shift in registry and thelattice constants of the matrix remain the same, the obstacle diametersare changed to create gaps, d, of different sizes, which are labeled onthe left side of the fluorescent image. The red bars on the fluorescenceprofile represent the width of the peaks (SD), and the black bars labelthe 1% inhomogeneity in the bead population a.u., arbitrary units.

FIG. 26 is a schematic illustrating the separation by deterministiclateral displacement in an array of microposts, with an example rowshift fraction of one-third. This shift creates three equal fluxstreamlines. The dashed lines are the boundaries between thestreamlines, which are assigned an index in the gaps between the posts.Paths of particles both smaller and larger than the critical thresholdare depicted with green and red dotted lines respectively. Smallparticles stay within a flow stream and large particles are displaced ateach obstacle. G is the clear spacing between the gap, is thecenter-to-center post separation, and d is the relative shift of thepost centers in adjacent rows.

These described methods allow for the quick and efficient formation ofuniformed sized droplet emulsion libraries for further use on amicrofluidic device of the present invention.

Example 2

The present invention provides methods for performing polymerase chainreaction (PCR). PCR can be performed on a drop-by-drop basis in amicrofluidic device according to the present invention. A monolithicchip can be provided wherein the heating and cooling lines are builtinto the chip and a sorting means is provided. Advantages of performingPCR in droplets on such a chip are that the chip is disposable and thereaction can be repeated without cleaning the device between reactions.Furthermore, the chip provides a convenient way of getting all thecomponents to perform PCR in the droplets in the right concentration.Additionally, the PCR is more efficient because the heat transfer ismore efficient due to the small volume. This provides for shorterincubation/residence 15 times. Droplets containing the nucleic acids,all PCR primers, and, if present, beads are generated one at a time atrates between 100 and 20,000 droplets per second. The droplets can thenbe sent through a serpentine path between heating and cooling lines toamplify the genetic material inside the droplets. Upon exiting thedevice the droplets may be sent for further on-chip or off-chipprocessing, directed into another chip, or the emulsion may be broken torelease the PCR product. If present, beads may be harvested by passingthe emulsion through a filtration device, sedimentation, orcentrifugation.

The width and depth of the channel can be adjusted to set the residencetime at each temperature, which can be controlled to anywhere betweenless than a second and minutes. At a typical rate of 1000 drops persecond, 1 million strands of DNA can be amplified in approximately 20minutes on one device. A typical flow rate of 250 μL/hour wouldcorrespond to 1000 drops of 50 microns in diameter being generated everysecond. Flow rates and droplet sizes can be adjusted as needed bycontrolling the nozzle geometry.

The present invention also provides methods for performingdideoxynucleotide sequencing reactions on a microfluidic device. Chainterminator sequencing (Sanger sequencing) is well known to those ofordinary skill in the art. DNA template preparation, cycling sequencingand preparing extension products for electrophoresis are relatedtechniques and also well known to those of skill in the art. AppliedBiosystems' “Automated DNA Sequencing: Chemistry Guide” 2000 is anexcellent resource covering these techniques and is incorporated hereinby reference in its entirety.

One method is to sequencing PCR templates which can include singleamplification PCR or nested and semi-nested PCR strategies. In thesimplest PCR sequencing case, the target DNA is amplified with a singleset of primers and then sequenced using the same primers. For manysamples, this works well. For the samples that do not work well withthis method, optimization of the PCR amplification may be required.Optimizing the PCR minimizes the presence of non-specific product bandsand ensures adequate yield. A single PCR amplification is alsocompatible with the use of a sequencing primer that binds internally(semi-nested or nested) to one or both of the PCR primers. This can behelpful if primer-dimer (primer oligomerization) artifacts are aproblem.

If difficulty with more complex samples, such as bacterial genomic DNA,is encountered a nested or semi-nested PCR can be used. These techniquesare useful when the target is present in small quantity. They offer morespecificity, which provides superior sequencing data with reducedbackground signal. Both nested and semi-nested PCR require twoamplifications. The first amplification is identical for nested andsemi-nested, but the second amplification differs as described below.Amplify with one set of PCR primers, which converts a complex sample(such as bacterial genomic DNA) into a non-complex sample consisting ofthe first PCR product and some side products. Nested PCR: Amplify 1% orless of the first PCR reaction product using a second set of PCR primersthat hybridize at positions internal to the first set. Semi-nested PCR:Only one primer of the second set of PCR primers is internal. The otherprimer is one of the original PCR primers.

A PCR primer can be synthesized with a universal sequencing primerbinding site added to the 5′ end (e.g., see Appendix E in AppliedBiosystems' “Automated DNA Sequencing: Chemistry Guide” for universalprimer sequences). This allows any PCR product to be sequenced withuniversal primers. Universal-tailed PCR primers enable the use ofcommercially available dye-labeled sequencing primers. This technique isalso useful with dye terminator chemistries, because universalsequencing primers have good annealing characteristics. However, thelonger PCR primers add to the overall cost of the reactions. Usinguniversal-tailed primers sometimes results in primer oligomerization. Asthese products have priming sites present, they can result in noisy datafor the first 20-100 bases. Redesigning the PCR primer, optimizing thePCR amplification further, and employing Hot Start methods can helpovercome this situation.

After PCR amplification, the resulting PCR product is in solution alongwith PCR primers, dNTPs, enzyme, and buffer components. The method usedto prepare the PCR product for sequencing depends on the amounts ofthese components that are carried over and on the chemistry used forsequencing. Excess PCR primers carried over from the amplificationreaction compete with the sequencing primer for binding sites andreagents in the sequencing reaction. This carryover of PCR primerspresents more of a problem in dye terminator chemistries because the dyelabel is incorporated into the extension product after the primeranneals to the template. If more than one primer is present, multipledye-labeled sequence ladders are generated, resulting in noisy data.Excess dNTPs from the amplification reaction can affect the balance ofthe sequencing reaction, resulting in decreased termination in shorterextension fragments.

Nonspecific PCR products include primer-dimer artifacts and secondaryPCR products. The presence of any significant quantity of either in aPCR product can result in poor quality sequencing data. Nonspecific PCRproducts behave as templates in the sequencing reaction and produceextension products, which results in noisy data. These products oftencan be visualized on an agarose gel before sequencing. If they arepresent, the PCR amplification should be optimized and repeated beforesequencing. Use of a nested or semi-nested sequencing primer can alsoallow good sequence data to be obtained. Alternatively, the PCR productof interest can be purified by agarose gel electrophoresis.

There are several ways to minimize contaminants in a PCR amplification:PCR optimization (Innis and Gelfand, 1990): (1) Amount of starting DNA;(2) Careful primer design; (3) Primer concentration, (4) Enzymeconcentration, (5) Magnesium ion (Mg2+) concentration, (6) Nucleotideconcentration; (7) Buffer composition; (8) Number of cycles; (9) pH;(10) Manual Hot Start method; (11) AmpliTaq Gold® DNA Polymerase as anautomatic Hot Start and/or (12) Limiting dNTPs and primers. All of thesemethods increase the specificity of the PCR amplification and decreasethe amount of contaminants that can interfere with a sequencingreaction.

There are several methods for purifying PCR products: (1) Columnpurification; (2) Ethanol precipitation; and/or (3) Gel purification.

An alternative to one of the more stringent purification methods listedabove is treatment of PCR products with shrimp alkaline phosphatase(SAP) and exonuclease I (Exo I) before sequencing. The SAP/Exo Iprocedure degrades nucleotides and single-stranded DNA (primers)remaining after PCR (Werle et al., 1994). This procedure is particularlyuseful in cases where limiting concentrations of primers and nucleotidescannot be used for direct PCR sequencing.

FIG. 27 shows one embodiment for a DNA sequencing chip design. TemplateDNA and primers are combined at step ‘add 1’ and the reaction isincubated at 95° C. for a hot start (position 1). The reaction thencycles 20-30 times (position 2) before the addition of SAP and ExoI at‘add 2.’ The reaction is incubated at 37° C. for a pre-definedtime-period and then the SAP and ExoI enzymes are inactivated at 95° C.(position ‘4’). The SAP/ExoI procedure degrades nucleotides andsingle-stranded DNA (primers) remaining after PCR. The universalsequencing primers, ddNTPs and buffers are added at ‘add 3,’ and the PCRsequencing reaction is allowed to proceed at position ‘5.’ The finalreaction product is collected and can be stored off-chip.

Step Action

1. For each sample, combine the following:

-   -   SAP(1 Unit/μL), 2 μL    -   Exo I (10 Units/μL), 0.2 μL    -   Deionized water, 6.0 μL    -   Note In general this procedure works well using 0.5 units of        each enzyme per microliter of PCR products used. The procedure        seems to work equally well with or without the use of SAP        buffer, so this has been excluded in this protocol.

2. Add 4.0 μL of PCR product to the above mix.

3. Incubate at 37° C. for 1 hour.

4. Incubate at 72° C. for 15 minutes to inactivate the enzymes.

The recommended DNA quantities for sequencing reactions are shown inTable 3-1 below.

TABLE 3-1 Recommended Ranges of DNA Template Quantity for Each ChemistryCycle Sequencing Chemistry Rhodamine Fluorescein/ Dye dRhodamine BigDyeRhodamine BigDye Template Terminator Terminator Terminator Dye PrimerPrimer PCR product:  100-200 bp   1-3 ng  1.-3 ng   1-3 ng   2-5 ng  2-5 ng  200-500 bp   3-10 ng   3-10 ng   3-10 ng   5-10 ng   5-10 ng 600-1000 bp   5-20 ng   5-20 ng   5-20 ng   10-20 ng   10-20 ng1000-2000   10-40 ng   10-40 ng   10-40 ng   20-50 ng   20-50 ng bp  40-100 ng   40-100 ng   40-100 ng   50-150 ng   50-150 ng >2000 bpsingle-  100-250 ng   50-100 ng   50-100 ng  150-300 ng  150-400 ngstranded     double-  200-500 ng  200-500 ng  200-500 ng  300-600 ng 200-800 ng stranded coamid, BAC  0.5-2.0 μg not  0.6-1.0 μg  0.5-2.0 μg 0.5-1.0 μg recommended bacterial not recommended   2-3 μg notrecommended genomic DNA

PCR protocols that limit amounts of primers and dNTPs allow the productof the reaction to be used for sequencing with no purification. This isusually carried out by setting up the PCR amplification with 5-10 pmolof primers and 20-40 μM dNTPs, so that most of the primers and dNTPs areexhausted during amplification. If the yield of the desired PCR productis high and the product is specific, i.e., it produces a single bandwhen analyzed by agarose gel electrophoresis, the sample can be dilutedbefore sequencing and will give good results. The dilution ratio dependson the concentration of your PCR product and needs to be determinedempirically (start with 1:2 and 1:10 dilutions with deionized water).When you limit concentrations of primers and dNTPs and dilute the PCRproducts, the PCR parameters have to be robust. Direct PCR sequencing ismost useful in applications where the same target is being amplified andsequenced repeatedly and PCR conditions have been optimized. Direct PCRsequencing can be done with dye primer chemistries. With dye terminatorchemistries, it is much more critical that the PCR primers be consumed.Excess PCR primers will be extended and labeled by the cycle sequencingreaction, resulting in noisy data. Direct PCR sequencing does not workfor XL PCR because limiting amounts of primers and dNTPs cannot be used.The PCR product should be purified or the excess primers and nucleotidesshould be degraded by SAP/Exo I treatment.

Example 3

The present invention provides methods for performing isothermal-typeamplification methods on a microfluidic device. Isothermal amplificationis an alternative to the standard PCR techniques described herein.Isothermal amplification is used to reduce the relative amount ofbackground DNA in a sample. Primers are generally used in a constanttemperature means of amplification. Isothermal amplification isapplicable for SNP detection. Once the DNA is amplified by isothermalamplification there are several well-known means for detecting whichnucleotide polymorphism is present. These include, but are not limitedto; allele specific primer extension, oligonucleotide ligation assay,mini-sequencing, fluorescence polarization, etc. Isothermalamplification is also applicable for DNA sequencing preparation. Theisothermally-amplified DNA can be attached to a solid phase within adroplet or placed within a parking space on chip. The beads or parkingspaces can be accessed and the amplified DNA used for a DNA sequencingreaction. Further, isothermal amplification is applicable for geneexpression analysis. Isothermal amplification can be used to monitorgene expression by the measurement of the amount of cDNA produced in aquantitative fashion. Many methods for isothermal amplification areknown in the art, including but not limited to the following examples.

Rolling circle amplification (RCA). A DNA polymerase extends a primer ona circular template, generating tandemly linked copies of thecomplementary sequence of the template (Fire & Xu, 1995). The TempliPhiamplification process using rolling circle amplification is known in theart. In the process, random hexamer primers anneal to the circulartemplate DNA at multiple sites. Phi29 DNA polymerase extends each ofthese primers. When the DNA polymerase reaches a downstream-extendedprimer, strand displacement synthesis occurs. The displaced strand isrendered single-stranded and available to be primed by more hexamerprimer. The process continues, resulting in exponential, isothermalamplification.

Transcription mediated amplification (TMA). An RNA polymerase is used tomake RNA from a promoter engineered in the primer region, a reversetranscriptase to produce complementary DNA from the RNA templates andRNase H to remove the RNA from cDNA (Guatelli et al, 1990).

Strand-displacement amplification (SDA). A restriction endonuclease isused to nick the unmodified strand of its target DNA and the action ofan exonuclease-deficient DNA polymerase to extend the 30 end at the nickand displace the downstream DNA strand (Walker et al, 1992).Strand-displacement amplification is known in the art.

Helicase-dependent amplification (HDA). A DNA helicase is used togenerate single-stranded templates for primer hybridization andsubsequent primer extension by a DNA polymerase. Schematic diagram ofHAD is shown in FIG. 28. Two complementary DNA strands are shown as twolines: the thick one is the top strand and the thin one is the bottomstrand. 1: A helicase (black 30 triangle) separates the twocomplementary DNA strands, which are bound by SSB (grey circles). 2:Primers (lines with arrow heads) hybridize to the target region on thessDNA template. 3: A DNA polymerase (squares with mosaic patterns)extends the primers hybridized on the template DNA. 4: Amplifiedproducts enter the next round of amplification.

One example is emulsion-based sample preparation. Random libraries ofDNA fragments are generated by shearing an entire genome and isolatingsingle DNA molecules by limiting dilution. See, FIG. 29. Specifically,sequencing reactions such as those performed by Solexa, 454 LifeSciences and others involve randomly fragmenting the entire genome,adding specialized common adapters to the fragments, capturing theindividual fragments on their own beads and, within the droplets of anemulsion, clonally amplifing the individual fragment (FIGS. 29 a, 29 b).Unlike in current sequencing technology, their approach does not requiresubcloning or the handling of individual clones; the templates arehandled in bulk within the emulsions. Typically, about 30% of the beadswill have DNA, producing 450,000 template-carrying beads per emulsionreaction.

Sample preparation and DNA sequencing is shown in FIG. 29. Panel A,Genomic DNA is isolated, fragmented, ligated to adapters and separatedinto single strands (top left). Fragments are bound to beads underconditions that favor one fragment per bead, the beads are captured inthe droplets of a PCR-reaction-mixture-in-oil emulsion and PCRamplification occurs within each droplet, resulting in beads eachcarrying ten million copies of a unique DNA template (top, second fromthe left). The emulsion is broken, the DNA strands are denatured, andbeads carrying single-stranded DNA clones are deposited into wells of afiber-optic slide (bottom left). Smaller beads carrying immobilizedenzymes required for pyrophosphate sequencing are deposited into eachwell (bottom, second from the left). Panel B, Microscope photograph ofemulsion showing droplets containing a bead and empty droplets. The thinarrow points to a 28-mm bead; the thick arrow points to an approximately100-mm droplet. Panel C, Scanning electron micrograph of a portion of afiber-optic slide, showing fiber-optic cladding and wells before beaddeposition. Panel D, The sequencing instrument consists of the followingmajor subsystems: a fluidic assembly. Panel E, a flow chamber thatincludes the well-containing fiber-optic slide. Panel F, a CCDcamera-based imaging assembly. Panel G, and a computer that provides thenecessary user interface and instrument control.

Another example is sequencing in fabricated picolitre-sized reactionvessels. One method uses sequencing by synthesis simultaneously in openwells of a fiber-optic slide using a modified pyrosequencing protocolthat is designed to take advantage of the small scale of the wells. Thefiber optic slides are manufactured by slicing of a fiber-optic blockthat is obtained by repeated drawing and fusing of optic fibers. At eachiteration, the diameters of the individual fibers decrease as they arehexagonally packed into bundles of increasing cross-sectional sizes.Each fiber-optic core is 44 μm in diameter and surrounded by 2-3 μm ofcladding; etching of each core creates reaction wells approximately 55μm in depth with a centre-to-centre distance of 50 μm (FIG. 29c ),resulting in a calculated well size of 75 pl and a well density of 480wells per square mm. The slide, containing approximately 1.6 millionwells, is loaded with beads and mounted in a flow chamber designed tocreate a 300-μm high channel, above the well openings, through which thesequencing reagents flow (FIG. 29d ). The unetched base of the slide isin optical contact with a second fiber optic imaging bundle bonded to acharge-coupled device (CCD) sensor, allowing the capture of emittedphotons from the bottom of each individual well (FIG. 29d ). Athree-bead system has been developed and the components optimized toachieve high efficiency on solid support. The combination ofpicoliter-sized wells, enzyme loading uniformity allowed by the smallbeads and enhanced solid support chemistry enabled users to develop amethod that extends the useful read length of sequencing-by-synthesis to100 bases.

In the flow chamber cyclically delivered reagents flow perpendicularlyto the wells. This configuration allows simultaneous extension reactionson template-carrying beads within the open wells and relies onconvective and diffusive transport to control the addition or removal ofreagents and by-products. The timescale for diffusion into and out ofthe wells is on the order of 10 s in the current configuration and isdependent on well depth and flow channel height. The timescales for thesignal-generating enzymatic reactions are on the order of 0.02-1.5 s.The current reaction is dominated by mass transport effects, andimprovements based on faster delivery of reagents are possible. Welldepth was selected on the basis of a number of competing requirements:(1) wells need to be deep enough for the DNA-carrying beads to remain inthe wells in the presence of convective transport past the wells; (2)they must be sufficiently deep to provide adequate isolation againstdiffusion of by-products from a well in which incorporation is takingplace to a well where no incorporation is occurring; and (3) they mustbe shallow enough to allow rapid diffusion of nucleotides into the wellsand rapid washing out of remaining nucleotides at the end of each flowcycle to enable high sequencing throughput and reduced reagent use.After the flow of each nucleotide, a wash containing a pyrase is used toensure that nucleotides do not remain in any well before the nextnucleotide being introduced.

Another example is base calling of individual reads. Nucleotideincorporation is detected by the associated release of inorganicpyrophosphate and the generation of photons. Wells containingtemplate-carrying beads are identified by detecting a knownfour-nucleotide ‘key’ sequence at the beginning of the read. Raw signalsare background-subtracted, normalized and corrected. The normalizedsignal intensity at each nucleotide flow, for a particular well,indicates the number of nucleotides, if any, that were incorporated.This linearity in signal is preserved to at least homopolymers of lengtheight. In sequencing by synthesis a very small number of templates oneach bead lose synchronism (that is, either get ahead of, or fallbehind, all other templates in sequence). The effect is primarily due toleftover nucleotides in a well (creating ‘carry forward’) or toincomplete extension. Typically, a carry forward rate of 1-2% and anincomplete extension rate of 0.1-0.3% is seen. Correction of theseshifts is essential because the loss of synchronism is a cumulativeeffect that degrades the quality of sequencing at longer read lengths.

Methods have demonstrated the simultaneous acquisition of hundreds ofthousands of sequence reads, 80-120 bases long, at 96% average accuracyin a single run of the instrument using a newly developed in vitrosample preparation methodology and sequencing technology. With Phred 20as a cutoff, they are able to show that their instrument is able toproduce over 47 million bases from test fragments and 25 million basesfrom genomic libraries. Recent work on the sequencing chemistry andalgorithms that correct for crosstalk between wells suggests that thesignal distributions will narrow, with an attendant reduction in errorsand increase in read lengths. In preliminary experiments with genomiclibraries that also include improvements in the emulsion protocol, oneis able to achieve, using 84 cycles, read lengths of 200 bases withaccuracies similar to those demonstrated here for 100 bases. Onoccasion, at 168 cycles, individual reads that are 100% accurate overgreater then 400 bases have been generated.

Isothermal amplification reactions, as described above, have shown greatpromise generating high yields with great fidelity. However, anassociated drawback arises from the tendency of the polymerase togenerate spurious, non-template amplification products when reactionsare conducted in the absence of template DNA. Additionally, ourapplication utilizes high microfluidic throughput in conjunction withlimiting DNA template dilutions to amplify single template molecules. Asa result, 10 the number of empty reaction droplets increasesconsiderably, comprising 90% or more of the total droplet populationfollowing Poisson distributions. Non-template amplification (hereafterNTA) in even a small fraction of the total droplets can confoundamplification detection strategies based on laser interrogation ofintercalating dyes, thus this issue must be resolved. To address thisproblem in the art, the present invention provides the use of mixedmodified and standard hexamer primers in microfluidic reactions toretard NTA while allowing template-based amplification to proceed.

Previous work has attempted to reduce NTA through incorporation ofnitroindole bases (Loakes and Brown 1994; Loakes, Hill et al. 1997) inthe random primers (Lage, Leamon et al. 2003) or reducing reactionvolumes to 600 nL (Hutchison, Smith et al. 2005). Unfortunately,modified nitroindole primers have proven difficult to replicate, andoften have the effect of significantly reducing the overall rate andyield of the amplifications in which they are incorporated. Low volumereactions conducted in multiplate wells have encountered difficultiesstemming from dispensation of low volumes, and associated issues ofsample evaporation, well to well contamination, etc.

The modified primers of the present invention containing nitroindolesand C3 non-replicable elements were studied in an effort to reduce NTAboth in bulk and microfluidic reactions. Both nitroindoles and C3non-replicable elements were found to be effective in reducing NTA, withprimers containing two 5′ nitroindoles most effective in NTAsuppression. However, increased NTA suppression was tightly linked withreduced yield in template amplification reactions. Amplifications usinga ratio of nitroindole to random hexamer primers generated a range ofboth template and non-template amplification yields, with a 15:85 ratioof nitroindole to random hexamers generating template yieldscommensurate with random hexamers primers alone, but generating littleif any spurious product in the absence of template.

Example 4

The PCR and isothermal amplifications described herein can be veryuseful in performing single nucleotide polymorphism analysis. A SingleNucleotide Polymorphism, or SNP, is a small genetic change, orvariation, that can occur within a person's DNA sequence. The geneticcode is specified by the four nucleotide “letters” A (adenine), C(cytosine), T (thymine), and G (guanine). SNP variation occurs when asingle nucleotide, such as an A, replaces one of the other threenucleotide letters—C, G, or T.

An example of a SNP is the alteration of the DNA segment AAGGTTA toATGGTTA, where the second “A” in the first snippet is replaced with a“T”. On average, SNPs occur in the human population more than 0.1percent of the time. Because only about 3 to 5 percent of a person's DNAsequence codes for the production of proteins, most SNPs are foundoutside of “coding sequences”. SNPs found within a coding sequence areof particular interest to researchers because they are more likely toalter the biological function of a protein. Because of the recentadvances in technology, coupled with the unique ability of these geneticvariations to facilitate gene identification, there has been a recentflurry of SNP discovery and detection.

Although many SNPs do not produce physical changes in people, scientistsbelieve that other SNPs may predispose people to disease and eveninfluence their response to drug regimens. Currently, there is no simpleway to determine how a patient will respond to a particular medication.A treatment proven effective in one patient may be ineffective inothers. Worse yet, some patients may experience an adverse immunologicreaction to a particular drug. Today, pharmaceutical companies arelimited to developing agents to which the “average” patient willrespond. As a result, many drugs that might benefit a small number ofpatients never make it to market.

In the future, the most appropriate drug for an individual could bedetermined in advance of treatment by analyzing a patient's SNP profile.The ability to target a drug to those individuals most likely tobenefit, referred to as “personalized medicine”, would allowpharmaceutical companies to bring many more drugs to market and allowdoctors to prescribe individualized therapies specific to a patient'sneeds.

Finding single nucleotide changes in the human genome seems like adaunting prospect, but over the last 20 years, biomedical researchershave developed a number of techniques that make it 25 possible to dojust that. Each technique uses a different method to compare selectedregions of a DNA sequence obtained from multiple individuals who share acommon trait. In each test, the result shows a physical difference inthe DNA samples only when a SNP is detected in one individual and not inthe other. Currently, existing SNPs are most easily studied usingmicroarrays. Microarrays allow the simultaneous testing of up tohundreds of thousands of separate SNPs and are quickly screened bycomputer.

The race among pharmaceutical companies today is to apply new systemgenomics approach to identify novel targets and validate these targetsin the most efficient fashion. SNP research will provide fundamentalunderstanding of many polygenic diseases, thus providing new therapeutictargets. As groups have performed genome-wide scans and other largestudies that require the genotyping of thousands of SNPs and samples, aneed for high-throughput SNP genotyping has become essential.

The microfluidic device of the present invention is capable ofperforming at least 10,000 SNP analysis per second, such that a fullgenome scan (i.e., 100K SNPs) with 10× overrepresentation can beperformed in less than an hour. The speed and efficiency permitted usingthe devices and methods of the present invention will significantlylower the associated costs and reagent consumption for performing SNPanalysis.

Example 5

The PCR and isothermal amplifications described herein can be veryuseful in providing necessary sample preparation processes forcommercial available DNA sequencers, such as the Solexa's 1G sequencer,which relies upon immobilized DNA for a substrate. In one embodiment,single molecules can be amplified using PCR primers treated withmoieties used for surface immobliziation, then flow the PCR positivedroplets across the surface of the slide, forming a packed emulsion. Theemulsion can then be broken, and the primers allowed to bind to theslide due the presence of the appropriate coating to bind the PCRproducts.

The Solexa 1G sequencer currently sequences amplified material that hasbeen amplified in place, on primers bound to the slide, by bridgeamplification. In this process, template DNA is flowed across the slidesurface at very low concentrations, and adapters previously ligated toeach template hybridize to complimentary primers attached to the slidein any one of eight lanes. Once hybridized, the primers are subjected tobridge amplification, a version of PCR amplification that utilizesimmobilized primers—the product of the reaction is a patch of DNAimmobilized to the slide. Several risks are encountered when amplifyingDNA in this matter—the process is exquisitely sensitive to DNAconcentration—if too much DNA is used, the DNA patches will be generatedtoo closely, or even overlap, generating mixed signals during subsequentsequencing. If too little DNA is used, the DNA patches will be presentat a very low density, and insufficient sequence may be generated duringthe run. As the sequencing reactions take 72 hours, and no titrationruns are conducted to test the DNA concentrations prior toamplification, the potential loss of time and money is considerable.Additionally, neither single molecule amplification nor bridgeamplification are very efficient, and bridge amplification has an upperand lower size limitation, generating products only within a particularlength range.

The present invention provides methods to overcome this limitations. Inone embodiment, a Solexa slide can be coated with any of the compoundscommonly used to permit binding and immobilization (e.g.,carboxy-esters, streptavidin, Igg, gold, etc.). PCR reactions could beperformed as described in the instant microfluidic device using primersmodified with the appropriate binding moiety (5′ amines, 5′ biotins, 5′DNPs or 5′ Thiols, respectively) to efficiently amplify PCR products insolution which could then be efficiently and easily bound to the Solexaslide for subsequent sequencing. The amplification is quite straightforward, conducted with a limiting dilution of template and a set ofprimer pairs compatible with the adapters ligated to the Solexatemplates through their standard sample preparation process. One of theprimer pairs would possess a 5′ binding moiety as described earlier,only one of the pair as this will permit removal of the opposing strandand the generation of single stranded immobilized templates on theslide. Once amplification has been conducted, and the positive dropletssorted, the droplets can be flowed onto each of the lanes of the Solexaslide. Proper spacing between the droplets can be obtained by mixingdroplets containing only buffer or immiscible oil in with the PCRpositive droplets at a ratio sufficient to ensure that the PCR positivedroplets are rarely proximal to each other. Once each lane has beenpacked with droplets, the droplets can be broken through the applicationof electrical field and the PCR products allowed to bind to the slide inthe same geographic area that the droplet had occupied. The immobilizedtemplates can be rendered single stranded through the application ofbasic washes, temperature etc. This will permit the rapid amplificationof PCR fragments and their subsequent density-controlled deposition ontothe Solexa chip for sequencing.

Example 6

The present invention provides methods for detecting the presence and/orsequence of nucleic acids in low copy number in droplets on amicrofluidic device. The detection of a specific nucleic acid sequencepresent in a sample by probing the sample with a complementary sequenceof nucleic acids is a well known technique. Nucleic acids are highlyspecific in binding to complementary nucleic acids and are thus usefulto determine whether a specific nucleic acid is present in a sample. Onemust know the sequence of the specific nucleic acid to be detected andthen construct a probe having a complementary nucleic acid sequence tothe specific nucleic acid sequence.

Since nucleic acid probes are highly specific, it is preferable in somesituations to probe the nucleic acid sequence itself rather than theprotein produced by the nucleic acid sequence. As a particular example,a diagnostic method based solely on protein detection would beunreliable for determining the presence of infectious particles ofhepatitis B virus, due to the presence of significant levels ofnon-infectious antigen particles which lack the DNA genome. In anotherexample, the various subtypes of human papilloma virus found in eitherpre-cancerous or benign cervical tumors can be distinguished only by theuse of nucleic acid probe hybridization. Also, the specific geneticmakeup of an AIDS virus makes it certain that an assay based on thepresence of an AIDS virus specific nucleic acid sequence would besuperior as a diagnostic.

The naturally-occurring high number of ribosomal RNA, up to 100,000copies per cell, has been used by GenProbe to facilitate diagnosis ofcertain bacterial pathogens, such as Legionella and Mycoplasma, usingnucleic acid probes. However, this strategy cannot be used withnon-cellular pathogens, such as viruses, or with probed nucleic acidsequences with low copy numbers. Copy number is a particular problemwith the development of a nucleic acid probe method for the detection ofAIDS virus, where the integrated provirus may be present in less thanone of ten thousand peripheral blood lymphocytes. Thus, if theparticular nucleic acid sequence suspected to be present in a samplecould be amplified, the copy number problem could be circumvented andprobe assays could be more readily used.

In a normal biological sample, containing only a few cells, andconsequently only a few copies of a particular gene, it is necessary toutilize an amplification process in order to overcome the 5 copy numberproblem.

One method to amplify is to ‘grow out’ the sample, that is, to arrangeconditions so that the living biological material present in the samplecan replicate itself. Replication could increase the quantity of nucleicacid sequences to detectable levels. In the food industry, for example,in order to test processed food for the food-poisoning bacteriaSalmonella, food samples must be incubated for a number of days toincrease the quantity of nucleic acid copy numbers. In clinical samples,pathogens must also be allowed to increase their number by growing outover some considerable time.

Current methods utilize a process in which a sample suspected ofcontaining a target DNA sequence is treated with oligonucleotide primerssuch that a primer extension product is synthesized which in turn servesas a template, resulting in amplification of the target a DNA sequence.The primer extension product is separated from the template using heatdenaturation. Current methods also include a process for amplifying atarget DNA sequence having two separate complementary strands. Theprocess includes treating the strands with primers to synthesizeextension products, separating the primer extension products from thetemplates, and in turn using the primer extension products as templates.

Both of the above methods require either manual or mechanicalparticipation and multi-step operations by the user in the amplificationprocess and are restricted to amplifying DNA only. The steps involved inthese methods require the user to heat the sample, cool the sample, addappropriate enzymes and then repeat the steps. The temperature changescause the enzymes to loose their activity. Hence, the user is requiredto repeatedly supplement the amplification mixture with aliquots ofappropriate enzymes during the amplification process.

The present invention provides methods for detecting the presence and/orsequence of nucleic acids in low copy number in droplets on amicrofluidic device. In one embodiment of the invention, anucleotide/peptide nucleic acid (pna) probe (oligo probe) can betethered such that when it binds to a template DNA molecule in low copynumber located inside an aqueous emulsion (i.e., droplet) it turns on oractivates an enzyme. By way of nonlimiting example, an alkalinephosphatase conjugate can be placed on one and of the low copy numbernucleotide, and an inhibitor to alkaline phosphatase on the other end(like a molecular beacon). When the oligo probe binds to the low copynumber template the inhibitor is removed from the enzyme and the enzymeturns over the substrate. The tethers can be a protein complementationassay wherein the binding of the oligo probe to the low copy numbertemplate causes the enzyme to be active.

Various other embodiments are described herein and should not beconsidered as limiting to the invention. Example 1: Taqman chews abeta-gal alpha protein attached at the 3′ end of an oligo probe therebyreleasing free alpha subunit to bind to omega fragment in solution.Example 2: Two oligo probes sit down on the low copy number templateadjacent to each other, thereby bringing two subunits of a proteincomplementing assay reagent together. Example 3: A Taqman-like enzymethat releases an active moiety can also be used. The active moiety caninclude, for example, an enzyme that becomes activated upon release fromthe oligo probe, or a fluorescent group that is quenched while tetheredto the oligo probe. The use of a double-strand specific nuclease thatwill chew up the probe only when the probe is bound, thereby releasingactive enzyme or fluorescent substrates. Example 4: The probe has afluorescent group attached such that the detected hybridization causesthe release of the fluorescent group a 1 a Taqman. Example 5: The probehas an inactive enzyme attached such that the detected hybridizationcauses the release of the active enzyme by a Taqman-type release.Example 6: The probe has an inactive complementing enzyme attached suchthat the detected hybridization causes the release of the active moietyof the enzyme to be able to complement. Example 7: Two probes haveinactive enzyme moieties attached such that the detected hybridizationcauses the complementation and activation of the enzyme. Example 8: Twoprobes come together and allow a Fluorescence Resonance Energy Transfer(FRET) reaction to occur. This would require a FRET-oligo library.Almost all SNP or transcriptional profiling method may be amenable tothis concept.

Many assays are conducted on microfluidic devices are including, but notlimited to, protein-protein, antibody-antigen, nucleic acid-protein,nucleic acid-nucleic acid, ligand-protein, ligand nucleic acid,ligand-ligand, eukaryotic or prokaryotic cell surface moiety-secondmoiety, the measurement of two or more receptors on the surface of aneukaryotic or prokaryotic cell, the development of three-hybrid typesystems using tandem fusions, interactor-cofactor, etc. Many other typesof interactions are also known that can be adapted to the system. Assaysincorporating complementation assays can be used in both proteomics andgenomics.

Currently, these assay methods require methods require>10K fluoresceinmolecules to detect interaction/binding. In an embodiment, the presentinvention provides methods which reduce the number of interactingproteins by amplifying the signal of molecules that do interact. Inpreferred embodiments of some assays, stable emulsions may not beneeded. An advantage is that we can eliminate surfactants andstabilizing additives which can affect protein activity within droplets.

Likewise, one could conceivably look for loss of complementation in anyof the above assays. This loss or gain of complementation can be afunction of physical (e.g., heat, light) or chemical additions to, orformulation of the droplets. These interactions include, but are notlimited to, protein-protein, antibody-antigen, nucleic acid-protein,nucleic acid-nucleic acid, ligand-protein, ligand-nucleic acid,ligand-ligand, eukaryotic or prokaryotic cell surface moiety-secondmoiety, the measurement of two or more receptors on the surface of aneukaryotic or prokaryotic cell, the development of three-hybrid typesystems using tandem fusions, interactor-cofactor, etc. Many other typesof interactions are also known that can be adapted to the system. Assaysincorporating complementation assays can be used in both proteomics andgenomics.

The amount of each interacting partner may be with-in drop quantifiableby genetically or chemically coupling a reporter molecule (e.g., a dyeor quantum dot, GFP protein) to one and or the other. Similarly, thecomplementation assay described for enzymatic amplification can use oneof several different complementing proteins such that the concentrationof each partner can be calculated within the droplet by using differentenzyme substrates added to the droplets at the same or differing time.Timing of substrate addition is not critical and one of skill in the artwould readily recognize that addition can be done at different times.‘Killing’ by various denaturants, protease, etc. is also within thepurview of the skilled artisan.

In an embodiment, complementation assays can be used to add a specificaddition to, for example, an IVT synthesized protein. As an example, thes-peptide of RNaseA and the S protein wherein the S peptide isgenetically fused to the IVT-generated Ab and the S protein, uponbinding to the S peptide activates the RNaseA activity and thereby stopsfurther IVT synthesis. Similarly, any two complementing interactors canbe used to generate an activity.

By quantifying the amount of interactors in a droplet it may be possibleto derive interaction kinetics (affinity and disassociation constants asexamples). In an embodiment, the present invention provides methodswhich can allow the quantification of proteins below that which can beseen by fluorescent spectroscopy in the absence of amplification.Alternatively, amplification may also not be 20 needed if one cangenetically add a quantified number of reporters to the end of amolecule. For example, genetically fusing 10 GFP proteins onto the endof a protein would thereby increase the fluorescence intensity 10-fold.Similarly, a series of small complementing moieties can be fused ontothe end of a protein and there obviate the need for long geneticfusions. As an example, a series of 10 s peptides spaced apart by alinker would be able to each ‘grab hold’ of an S protein to generate anincreased signal over that which can be achieved by a single enzyme.Other examples include either biotin or biotin-binding protein mimeticand streptavidin or avidin, FLAG tags, poly histidines, complementingGFP, etc. Another example includes Qcoding the droplets.

FIGS. 30 and 31 show the current method for isolating antibodies on amicrofluidic device. In the current method, DNA beads are made usingbulk emulsion PCR; DNA-containing beads preferably isolated beforeloading onto a microfluidic device. The antigen can be <20 kD,(preferably<10 kD) and labeled with an appropriate dye, (preferably withseveral dye-molecules). It may even be preferable to put a poly-lysinetail onto the antigen to increase signal (but be concerned aboutquenching). The antigen is formulated into the bead-containing dropletsat a concentration of 100 nm to allow proper sensitivity in the droplet(use of multiple/different dyes may allow this concentration to drop).In some situations it is possible to add antigen at the same time as theIVT solution.

FIG. 32 shows the method of the present invention for isolatingantibodies on the microfluidic device. The right panel is a diagram ofindividual steps proposed to amplify signal of interacting antibody andantigen. The left panel is a schematic as would be designed for a chipto be used on microfluidic device.

FIG. 33 shows the genetic selection for full length antibody clones. Agenetic selection can be used to enrich for full-length antibody clonesby transforming E.coli and selecting for clones able 5 to grow on mediumin which a suitable sugar is the only carbon source.

Example 7

The present invention provides a microfluidic device topology andimplementation that merges the functionality of a first microfluidicsubstrate with that of a second microfluidic substrate by using forcedwithdrawal and reinjection into the same fluidic port. In currentmulti-step assays, the following steps must be performed to complete a“two step” experiment: (1) Installation of the first microfluidicsubstrate; (2) Installation of all first stage reagents; (3) Priming ofall fluid lines and stabilization of the device operation; (4) Passiveconnection of a storage container to the instrument after device hasbeen stabilized; (5) Disconnection of the storage container from thefirst device when collection complete; (6) Incubation of the collectedemulsion; (7) Removal of the first device from the instrument; (8)Cleaning of the fluid lines needed for the second step of theexperiment; (9) Installation of a second device to the instrument; (10)Connection of all second stage reagents, including the emulsioncollected during the first stage; (11) Priming of the fluidicconnections to the second device prior to running the second half of themeasurement; and (12) Collection/readout of the second stage.

The methods of the present invention replaces current methodology ofmulti-step assays with the following: (1) Installation of the firstmicrofluidic substrate; (2) Installation of all first and second stagereagents, including the first stage storage container (if it is notalready connected); (3) Priming and stabilization of the fluidic linesand device; (4) Controlled collection of the first stage combineddroplets by actively withdrawing some fraction of the oil and all of thegenerated emulsion into the storage container; (5) Incubation of thecollected emulsion; (6) Startup and reinjection of the collecteddroplets back into the second half of the device; and (8)Collection/readout of the second stage. FIG. 34 depicts a schematicrepresentation of this device topology.

Elimination of the handling of the collected emulsion has significantbenefits beyond simplifying the user interaction with the instrument.Contaminants have the potential to ruin the experiment, and any extrahandling and connection/disconnection increase the probability thatcontaminants will be introduced into the instrument.

Example 8

Gene silencing through RNAi (RNA-interference) by use of shortinterfering RNA (siRNA) has emerged as a powerful tool for molecularbiology and holds the potential to be used for therapeutic genesilencing. Short hairpin RNA (shRNA) transcribed from small DNA plasmidswithin the target cell has also been shown to mediate stable genesilencing and achieve gene knockdown at levels comparable to thoseobtained by transfection with chemically synthesized siRNA (T. R.Brummelkamp, R. Bernards, R. Agami, Science 296, 550 (2002), P. J.Paddison, A. A. Caudiy, G. J. Hannon, PNAS 99, 1443 (2002)). Possibleapplications of RNAi for therapeutic purposes are extensive and includesilencing and knockdown of disease genes such as oncogenes or viralgenes.

Many assays are conducted on microfluidic devices are including, but notlimited to, protein-protein, antibody-antigen, nucleic acid-protein,nucleic acid-nucleic acid, ligand-protein, ligand-nucleic acid,ligand-ligand, eukaryotic or prokaryotic cell surface moiety-secondmoiety, the measurement of two or more receptors on the surface of aneukaryotic or prokaryotic cell, the development of three-hybrid typesystems using tandem fusions, interactor-cofactor, etc. Many other typesof interactions are also known that can be adapted to the system.However, there is a need in the art for improved methods of RNAiscreening, quickly and accurately.

The present invention provides methods for the screening of lethal andsynthetic lethal RNAi-induced phenotype on a microfluidic device. Thepresent invention utilizes a lentiviral library of RNAi where each virushas a unique 60-nt identifying barcode bracketed on either side withnucleotide sequences common to all vectors.

The analysis of lethal and synthetic lethal RNAi-induced phenotypesoccurs in two steps. In the first step, the viral library is combined inbulk and infected, also in bulk, into an appropriate host strain. Themolar amount of each of the different lentivirus in the library ispre-determined by sequencing on, for example, an appropriate instrumentor by gene expression analysis on a microfluidic device. Post infection,the treated cells are collected and the 60-nt barcode is amplified fromchromosomal DNA using PCR primers based on the bracketing sequence. Inthe second step, the PCR amplification product is added to amicrofluidic device and analyzed against a labeled droplet librarywherein the labeled droplets contain lentiviral-barcode-quantificationreagents (e.g., molecular beacons, Taqman probes, etc.) against each ofsaid lentiviral barcodes. A gene-expression analysis-like analysis isperformed to quantify the amount of each lentiviral barcode-type in thetreated cells. An absence or significant decrease of any lentiviralbarcode in the amplified product can be assumed to be due to the deathof that barcode-containing lentivirus in the treated cells. In anembodiment, the products within the droplets can also be amplified.

GFP or additional transcription analysis can also occur in two steps. Instep one, the viral library is combined in bulk and infected, also inbulk, into an appropriate host strain. The molar amount of each of thedifferent lentivirus in the library is pre-determined by sequencing on,for example, an appropriate instrument or by gene expression analysis ona microfluidic device. Post infection, the treated cells are i)collected in bulk, ii) sorted using a phenotype able to be sorted in amicrofluidic device (e.g, GFP expression, cell-surface marker, low-copycell-surface marker, etc.) and iii) the 60-nt barcode is amplified fromchromosomal DNA using PCR primers based on the bracketing sequence. Inthe second step, the PCR amplification product is added to a microfludicdevice and analyzed against a labeled droplet library wherein thelabeled droplets contain lentiviral-barcode-quantification reagents(e.g., molecular beacons, Taqman probes, etc.) against each of saidlentiviral barcodes. A gene-expression analysis-like analysis isperformed to quantify the amount of each lentiviral barcode-type in thetreated cells. An absence or significant decrease of any lentiviralbarcode in the amplified product can be assumed to be due to the deathof that barcode-containing lentivirus in the treated cells.

Example 9

Many diseases are associated with particular chromosomal abnormalities.For example, chromosomes in cancerous cells frequently exhibitaberrations called translocations, where a piece of one chromosomebreaks off and attaches to the end of another chromosome. Identifyingsuch chromosome abnormalities and determining their role in disease isan important step in developing new methods for diagnosing many geneticdisorders. Traditional karyotyping using Giemsa staining allowsscientists to view the full set of human chromosomes in black and white,a technique that is useful for observing the number and size of thechromosomes. However, there is a need in the art for improved methods ofkaryotyping, quickly and accurately.

The present invention provides methods for karyotyping. Preferably, thekarotyping screens occur within droplets on a microfluidic device.Currently, scientists cannot accurately identify many translocations orother abnormalities using only a black and white karyotype. Spectralkaryotyping (SKY) is a laboratory technique that allows scientists tovisualize all 23 pairs of human chromosomes at one time, with each pairof chromosomes painted in a different fluorescent color. By using SKY,they can easily see instances where a chromosome, painted in one color,has a small piece of a different chromosome, painted in another color,attached to it.

Through the use of droplet-based methods, chromosomes can be capturedwithin droplets without having to worry about shear-forces. Thechromosomes can then be passed through a ‘neck down’ to stretch themout. Labeling prior to loading, with either Giemsa stain oroligonucleotide probes, can be used to karyotype the DNA as it flows.

The present invention provides methods of using SKY probes to ‘paint’individual chromosomes. Also provided is a method used by flowcytometrists for the preparation of chromosomes prior to flow analysis,including flow sorting. The present invention provides methods whichallow the adaptation of these methods for use on a microfluidic device.

SKY involves the preparation of a large collection of short sequences ofsingle-stranded DNA called probes. Each of the individual probes in thisDNA library is complementary to a unique region of one chromosome;together, all of the probes make up a network of DNA that iscomplementary to all of the chromosomes within the human genome. Eachprobe is labeled with a fluorescent molecule that corresponds to thechromosome to which it is complementary. For example, probes that arecomplementary to chromosome 1 are labeled with yellow molecules, whilethose that are complementary to chromosome 2 are labeled with redmolecules, and so on. When these probes are mixed with the chromosomesfrom a human cell, the probes hybridize, or bind, to the DNA in thechromosomes. As they hybridize, the fluorescent probes essentially paintthe set of chromosomes in a rainbow of colors. Scientists can then usecomputers to analyze the painted chromosomes to determine whether any ofthem exhibit translocations or other structural abnormalities. See, FIG.35.

Prior to analysis chromosomes can be prepared as described in Bee LingNg and Nigel P. Carter “Factors Affecting Flow Karyotype Resolution.Cytometry” Part A 69A:1028-1036 (2006) as follows:

-   -   1. Arrest cells at metaphase using 0.1 1 g/ml demecolcine for        optimal amount of time, dependent on the cell cycle time of the        cell lines. (Approximately 5 h for suspension, 16 h for adherent        cell lines and 4 h for LPS stimulated B lymphocyte culture).    -   2. Harvest cells and centrifuge at 289 g for 5 min. Remove        supernatant.    -   3. Resuspend cell pellet in 5 ml of hypotonic solution (75 mM        KCl, 10 mM MgSO4, 0.2 mM spermine, 0.5 mM spermidine, pH 8.0)        and incubate at room temperature for 10 min.    -   4. Centrifuge cell suspension at 289 g for 5 min. Remove        supernatant.    -   5. Resuspend cell pellet in 3 ml of ice cold polyamine isolation        buffer (PAB, containing 15 mM 20 Tris, 2 mM EDTA, 0.5 mM EGTA,        80 mM KCl 3 mM dithiothreitol, 0.25% Triton X-100, 0.2 mM        spermine, 0.5 mM spermidine, pH 7.50) and vortex for 20 s.    -   6. Briefly centrifuge chromosome suspensions at 201 g for 2 min.        Filter supernatant through 20 1 m mesh filter.    -   7. Stain chromosomes overnight with 5 1 g/ml of Hoechst, 401        g/ml chromomycin A3 and 10 mM MgSO4.    -   8. To the stained chromosome suspension, add 10 mM of sodium        citrate and 25 mM of sodium sulphite 1 h before flow analysis.

The present invention also provides methods of sorting chromosomes forkaryotyping wherein individual chromosomes are sorted. This sorting canbe performed after chromosome-specific identification (such ashybridization of labeled probes) so as to enrich a population for one ormore specific chromosomes. This enriched population can be used in DNAsequencing reactions.

The Giemsa-stained and/or labeled-probe-hybridized chromosomes can besent through a constriction of a channel on a microfluidic device todetect the areas of stain and/or label as a genetic ‘bar-code’ toidentify regions of translocation, etc. on individual chromosomes.

Identification of chromosomes and karyotyping can be used afterenrichment of specific cell-types, for example i) fetal cells frommaternal blood, or ii) cancer cells from human blood.

Example 10

The present invention provides microbial strains with improved biomassconversion and methods of preparing such strains. Biomass is organicmatter such as plant matter, i.e., trees, grasses, agricultural crops,or other biological material such as animal material. It can be used asa solid fuel, or converted into liquid or gaseous forms, for theproduction of electric power, heat, chemicals, or fuels. Biomass canalso be used in formulating other commercial products in otherindustrial sectors such as textiles, food supply, environmental,communication, housing, etc. For example, biofuel development seeks thedevelopment of new microbial strains with improved biomass conversion toethanol.

Researchers have been applying sophisticated metabolic engineeringtechniques to develop microorganisms that can more effectively fermentthe sugars in biomass. Lignocellulosic biomass contains five carbonsugars such as xylose (from the hemicellulose) as well as the more“common” six carbon sugars such as glucose found in grains. This makesfermentation and other bioprocessing far more challenging. While somebiorefinery scenarios will take advantage of the different sugar streamsto produce multiple products, others will be more cost effective if allthe sugars can coferment in a single set of equipment. Accordingly,researchers are developing microorganisms that can coferment all thesugars in biomass in order to improve ethanol production economics. Withindustrial partners, researchers are working to develop designer strainsof microorganisms for biomass conversion of specific feedstocks,feedstreams, and processes. Thus, there is a need for devices andmethods for the rapid engineering of new microbial strains with improvedbiomass conversion.

The present invention provides a microfluidic device in which toformulate a mutant bacterial, yeast, or fungi strain which can be usedfor biomass energy conversion. The microorganism strain can beengineered, e.g., by recombinant methods, to include at least onenucleic acid sequence encoding one or more polypeptides of interest,wherein the mutant strain expresses the polypeptides of interest at ahigher level than the corresponding non-mutant strain under the sameconditions.

In one embodiment, the nucleic acid sequence can be operably linked toan expression-regulating region selected from the group consisting of apromoter sequence associated with cellulase expression, xylanaseexpression, or gpdA expression. In another embodiment, the nucleic acidsequence can further be optionally linked to a secretion signalsequence.

In one embodiment, the nucleic acid sequence can be a heterologousnucleic acid sequence selected from heterologous polypeptide-encodingnucleic acid sequences, heterologous signal sequences, or heterologousexpression-regulating sequences, or combinations thereof. For instance,the nucleic acid sequence can be a heterologous signal sequence, e.g., asecretion signal sequence. Alternatively, the nucleic acid sequence canbe a heterologous expression-regulating region, e.g., an induciblepromoter or a high expression promoter.

The polypeptides of interest can be homologous peptides and areexpressed in the mutant strain at a higher level than in thecorresponding non-mutant strain under the same conditions. In oneembodiment, the polypeptides of interest can be selected from one ormore of carbohydrate-degrading enzymes, proteases, lipases, esterases,other hydrolases, oxidoreductases, and transferases. In yet anotherembodiment, the polypeptides of interest can be selected from one ormore of fungal enzymes that allow production or overproduction ofprimary metabolites, organic acids, secondary metabolites, andantibiotics. These fungal sequences can include secretion signalsequences, for example, and can be selected from one or more ofcellulase, β-galactosidase, xylanase, pectinase, esterase, protease,amylase, polygalacturonase or hydrophobin. Alternatively, the fungalsequences can include one or more fungal expression-regulating regions.Preferably, the polypeptides of interest exhibit optimal activity and/orstability at a pH above 6, and/or have more than 70% of its activityand/or stability at a PH above 6.

The mutant microorganism can further include a selectable marker. Theselectable marker can confer resistance to a drug, for example, orrelieve a nutritional defect.

In another embodiment, the microorganism can be mutated via mutagenesis.Mutagenesis can be achieved, for example, by one or both of UVirradiation or chemical mutagenesis. For example, in one embodimentmutagenesis can include exposing a microorganism to UV irraditation,exposing it to N-methyl-N′-nitro-N-nitrosoguanidine, and exposing itagain to UV irradiation.

The present invention also provides methods for creating microbialstrains with improved biomass conversion. In one embodiment, the methodincludes providing a microfluidic device made of a microfabricatedsubstrate. The microfabricated substrate can have a plurality ofelectrically addressable, channel bearing modules integrally arranged soas to be in fluid communication with each other, thereby forming atleast one main channel adapted to carry at least one continuous phasefluid. The method further includes flowing a buffer, a microbe library,and a media (either in separate solutions or all together in onesolution) through a first inlet channel into the main channel of themicrofabricated substrate such that one or more droplets is formed inthe continuous phase fluid; flowing a substrate through a second inletchannel into the main channel of the microfabricated substrate such thatone or more droplets is formed in said continuous phase fluid;coalescing the droplets containing the microbe library with the dropletscontaining the substrate as the droplets pass through a coalescencemodule, thereby producing a NanoRefinery; interrogating the NanoRefineryfor a predetermined characteristic within a detection module on themicrofabricated substrate; and collecting the NanoRefineries containingthe microbes of interest in a collection module on the microfabricatedsubstrate. An assay system, e.g., a means by which to determine whethera desired product has been produced, or a means by which to determinethe absence of a starting substrate material, can also be incorporatedinto one of the droplets or the NanoRefinery. The assay system can beadded either before, after, or simultaneously with the addition of thesubstrate.

In one embodiment, the assay system is a dye that can measure the amountof sugar in a solution. In another embodiment, the microbe library caninclude one or more of DNA, bacteria, yeast or fungi. The substrate caninclude biomass, which can include one or more of fermentation broth,cellulose or other polysaccharide, or plant lignan.

The present invention further provides methods for degrading orconverting biomass into one or more products. In one embodiment, themethod includes treating the biomass with an effective amount of arecombinant microorganism, wherein the recombinant microorganismexpresses or overexpresses one or more heterologous sequences encodingenzymes that degrade or convert the biomass into one or more products.In one embodiment, the biomass can include plant cell wallpolysaccharides. In another embodiment, the products can include one ormore of the commodity chemicals or secondary commodity chemicals used toproduce one or more of the Intermediates or finished products andconsumer goods listed in FIGS. 36 and 37.

Example 11

The present invention provides methods of screening for enzymes withimproved activity. As an example, at least one cell (prokaryotic oreukaryotic) is deposited into a droplet and the cells are allowed tosecrete a substance for which a homogeneous assay is available. For aspecific example, an individual Bacillus subtilis cell from amutagen-treated culture is deposited within a 30 micron growth-mediumcontaining droplet, the resulting droplets are collected and allowed toincubate overnight. The bacterium secretes a protease into the droplet.The droplets containing the Bacilli are then individually merged with anassay droplet containing a protease-cleavable dye-labeled peptide. Theuncleaved peptide is colorless, while the cleaved peptide becomes red.The droplets are 15 incubated on chip for a sufficient time such as toallow color formation. Droplets that are red are sorted. The collectedsorted droplets are then plated onto solid growth medium and theresulting colonies, after overnight incubation, represent individualclonal isolates from the droplets.

In addition, the assay droplets can be sorted based upon a specificactivity, for example enzymes that are more active, indicated by a moreintense red droplet after a specific period of time. In addition, theconditions within the droplet can be changed during the merging of thetwo droplets to assay conditions which may itself not be permissive forthe bacteria. For example the pH within the droplet is altered in orderto find mutant enzymes that work better under either acidic or alkalineconditions. Or the droplets can be heated such that enzymes that aremore heat resistant are identified.

The merging of the droplets can be right after the individual cell isplaced into the droplet or after further incubation.

Another method of the instant invention is as described above, exceptthat the cells are lysed before the assay step in order to release thecontents of the cell into the droplet.

In other embodiments, the substrate can be added with—or simultaneouslyformulated from two or more separate reagent streams with the containedwithin a droplet. For a specific example, macrophage cells are washed inbuffer and incubated with an enzyme-conjugated anti-cell-surfaceantibody. The cells are then individually loaded into dropletsformulated at the same time, with the enzyme substrate. The amount ofenzyme substrate turned over within the droplet will be proportional tothe number of enzyme molecules within the droplet, which is proportionalto the number of antibodies bound to the macrophage surface. By carefulcalibration it should be possible to estimate the number of cell-surfacemolecules attached to the cell surface.

What is claimed is:
 1. A method for preparing nucleic acids forsequencing on a chip, the method comprising: providing a plurality ofnucleic acid fragments and a plurality of microbeads; encapsulating theplurality of nucleic acids fragments and the plurality of microbeads inan emulsion comprising droplets, wherein each droplet contains onenucleic acid and one microbead; amplifying the nucleic acids fragments,wherein the amplifying also generates bead-bound nucleic acid fragments;breaking the emulsion; and depositing the bead-bound nucleic acidfragments onto a chip for sequencing.
 2. The method of claim 1, whereinthe nucleic acid comprises DNA or RNA.
 3. The method of claim 1, whereinthe nucleic acid comprises an adaptor.
 4. The method of claim 1, whereinthe droplets comprise primers.
 5. The method of claim 4, wherein theprimers are coupled to the microbeads.
 6. The method of claim 1, furthercomprising shearing the nucleic acid.
 7. The method of claim 1, whereinthe nucleic acid comprises an entire genome.
 8. The method of claim 1,wherein the droplets are surrounded by an immiscible carrier fluid. 9.The method of claim 1, wherein the amplification reaction comprises apolymerase chain reaction.
 10. The method of claim 1, wherein theamplification reaction comprises flowing the droplets through amicrofluidic device to expose the droplets to thermocycling.
 11. Themethod of claim 1, wherein the chip comprises a plurality of wells. 12.The method of claim 11, wherein each of the bead-bound nucleic acidfragments is deposited into a well.
 13. The method of claim 1, furthercomprising detecting droplets comprising bead-bound nucleic acidfragments.
 14. The method of claim 13, further comprising sorting thedroplets comprising bead-bound nucleic acid fragments from droplets thatdo not comprise bead-bound nucleic acid fragments.